Genetic Screening of Tuberous Sclerosis Complex in Sicily with a Focus on Neurological Manifestations

Genetic Screening of Tuberous Sclerosis Complex in Sicily with a Focus on Neurological Manifestations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Genetic Screening of Tuberous Sclerosis Complex in Sicily with a Focus on Neurological Manifestations Andrea Domenico Pratico', Claudia Di Napoli, Stefania Salafia, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5804009/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder characterized by widespread hamartomas in multiple organs and significant neurological involvement. TSC is caused by pathogenic variants in TSC1 or TSC2 genes, leading to hyperactivation of the mTOR pathway and consequent dysregulation of cell growth. These tumor suppressor genes encode hamartin and tuberin, proteins critical for regulating cell proliferation, neuronal excitability and synaptogenesis. In this retrospective study, we analyzed clinical, genetic and radiological features of 81 TSC patients from Sicily, focusing on genotype-phenotype correlations and intergroup comparisons. TSC2 mutations were more common than TSC1 mutations (61.7% vs. 38.3%). Patients with TSC2 mutations Patients with TSC2 mutations tended to exhibit a higher frequency of weekly seizures, a higher prevalence of infantile spasms and hypsarrhythmia compared to those with TSC1 mutations, consistent with a more severe phenotype. Interestingly, TSC1 patients exhibited a higher incidence of radial bands, while TSC2 patients harbored a larger average size of tubers and subependymal nodules. Cognitive and behavioral disorders were similarly distributed, although TSC1 patients had higher rates of normal or borderline cognitive function, while TSC2 patients had more severe neuropsychiatric profiles compared to TSC1. Additionally, we present four novel potential genotype-phenotype correlations. To our knowledge, these is the first comprehensive TSC1 and TSC2 mutational analysis and genotype-phenotype correlation study carried out in in a large cohort of Sicilian patients affected by TSC. Our findings contribute to regional and global data on TSC, emphasizing the utility of genotype-informed management strategies. Health sciences/Neurology/Neurological disorders/Paediatric neurological disorders Biological sciences/Genetics/Clinical genetics TSC1 TSC2 Tuberous Sclerosis Complex Seizures Genotype/Phenotype Figures Figure 1 INTRODUCTION Tuberous Sclerosis Complex (TSC) (OMIM #191100; #613254) is a rare genetic disorder affecting approximately 1:6000-1:10,000 live births [ 1 ]. First described by de Bourneville, TSC manifests with a wide clinical spectrum, including cutaneous, cardiac, renal, and neurological involvement, alongside neuropsychiatric manifestations known as TAND (Tuberous Sclerosis-Associated Neuropsychiatric Disorders) [ 2 , 3 ]. The disease is caused by pathogenic heterozygous mutations in either the TSC1 gene (OMIM *605284) on chromosome 9 or the TSC2 gene (OMIM *191092) on chromosome 16 [ 4 , 5 ]. About two-thirds of cases result from de novo pathogenic variants, especially for the TSC2 gene, while familial cases demonstrate equal distribution of variants between the two genes. Notably, 7.4% of patients meeting clinical diagnostic criteria lack identifiable mutations in TSC1 or TSC2 through conventional genetic testing, with somatic mosaicism and intronic mutations implicated in these cases [ 6 , 7 ]. The hamartin-tuberin complex, encoded by TSC1 and TSC2 , acts as a negative regulator of the mechanistic target of rapamycin (mTOR) pathway. Dysfunction in this complex due to inactivating mutations leads to constitutive mTOR activation, resulting in cellular hyperproliferation and aberrant growth [ 8 ]. Clinical diagnostic criteria for TSC, first established in 2012 and revised in 2021, now encompass 11 major and seven minor features [ 9 ]. Histopathologically, TSC is marked by benign tumors (hamartomas) in multiple organs and cortical brain lesions termed "tubers", which disrupt synaptic networks and contribute to the disorder's neurological manifestations. Despite nearly complete penetrance, the clinical presentation of TSC is highly variable. Neurological symptoms such as epilepsy, cognitive impairment, and autism spectrum disorder (ASD) are predominant and significantly impact patients’ quality of life [ 10 , 11 ]. Recent studies, including data from the TOSCA registry, consistently show that TSC2 mutations are associated with more severe clinical phenotype than TSC1 mutations [ 9 , 12 ]. This study analyzes the genotype-phenotype relationships in a Sicilian cohort, and evaluates the alignment of these findings with global research, with particular regard to epilepsy and neurological comorbidities. PATIENTS AND METHODS Patients and Clinical Assessment This study included 81 patients (mean age 26 years, range 2–71) from four Italian centers (Unit of Pediatric Clinic and Unit of Rare Diseases of the Nervous System in Pediatric Age – "Policlinico-San Marco” University Hospital, Catania; Clinical and Instrumental Neurology and Neurophysiopathology Unit – I.R.C.C.S. Oasi Troina; Regional Reference Center for Rare Genetic and Chromosomal Diseases – Maternal and Child Department, "Villa Sofia-Cervello" Hospital, Palermo; Child Neuropsychiatry Unit, Santa Marta and Santa Venera Hospital, Acireale). Patients must present with a definite clinical diagnosis of TSC and a confirmed genetic alteration in TSC1 or TSC2 , including pathogenic and likely pathogenic variants, encompassing exonic mutations. Patients harboring intronic mutations with unknown or unpredictable effects at the protein level were included only if they had a definitive diagnosis of TSC, established according to the revised 2021 diagnostic criteria. [ 9 ]. Retrospective analysis used data from existing medical records, supplemented by additional studies and follow-up during the period of the study. Collected data covered clinical, genetic, and radiological information. Regarding the epilepsy phenotype, for each patient data included age at onset (in months), seizure type, frequency (seizures per week, averaged over the most recent 12-month period), EEG findings, and treatment history. Seizure outcome was categorized based on response to treatment as follows: remission (> 75% reduction in seizure frequency), good control (50–75%), partial control (25–50%), and drug-resistant epilepsy (< 25%). Data were obtained through clinical records and follow-up evaluations. Classifications were made in accordance with standard clinical definitions. All patients had performed in the last three years an EEG, a brain MRI and a neuropsychiatric evaluation. Clinical investigation included a review of medical history and a careful physical examination by clinicians experienced in the manifestations of TSC. Brain MRI reports were collected and analyzed to identify specific patterns associated with cortical tubers, subependymal nodules (SENs), sub-ependymal giant cell astrocytoma (SEGA), and radial bands. Details on clinical signs and symptoms were obtained from the physicians who were sent a standardized clinical evaluation form. Members of the family were consulted when necessary. In addition, a range of standardized and validated psychological measures were used to assess intellectual level, autistic disorder, anxiety, depression, and behavior disorders. All manifestations were graded according to a binary scale (present or absent), but several features were graded in severity using numbers and words (characteristics of seizure, drug resistance, severity of intellectual disability, for example). Although complete phenotype information was collected, neurological and neuropsychiatric characteristics were the primary focus of analysis (supplementary tables 1 and 2). All TSC patients included in the study provided informed consent and had molecular diagnostic analysis for variants in TSC1 or TSC2 . The study protocol was approved by the Ethical Committee of the University of Catania, Italy. All methods were performed in accordance with the relevant guidelines and regulations Mutational Analysis Genetic analysis was performed to identify pathogenic variants in TSC1 and TSC2 using a combination of multiplex ligation-dependent probe amplification (MLPA), denaturing high-performance liquid chromatography (DHPLC), and next-generation sequencing (NGS). MLPA (MRC-Holland, SALSA P124/P046) was used to detect large deletions or duplications in TSC1 and TSC2 , while DHPLC was initially employed as a screening method to identify potential sequence variants, which were subsequently confirmed by Sanger sequencing. Next-generation sequencing (NGS) was the preferred approach for patients born after 2016, allowing for a more comprehensive mutation screening. Targeted NGS was performed using a custom gene panel covering the entire coding regions and exon-intron boundaries of TSC1 (9q34.13) and TSC2 (16p13.3). The target region encompassed all exons and at least 20 bp of flanking intronic sequences to detect splicing-affecting variants. Sequencing was conducted on the Illumina MiSeq/NextSeq platform, with a mean coverage of > 200×, ensuring a minimum of 30× for reliable variant calling. Raw sequence reads were processed using BWA-MEM for alignment to the hg19/hg38 reference genome, and variant calling was performed using GATK HaplotypeCaller. Identified variants were annotated using ANNOVAR, with pathogenicity assessed based on ClinVar, gnomAD, and ACMG guidelines. Large genomic deletions or duplications not detected by NGS were confirmed by MLPA. Patients without identified pathogenic variants in TSC1 or TSC2 and not meeting the diagnostic criteria for Tuberous Sclerosis Complex were excluded from genotype-phenotype correlations. Neuropsychological evaluation Neuropsychiatric characteristics and cognitive impairment were evaluated using standardized neuropsychological tests administered by trained clinicians. Cognitive functioning was assessed using the Wechsler Preschool and Primary Scale of Intelligence (WPPSI-III or WPPSI-IV) for children aged 2 years to 7 years, and Wechsler Intelligence Scale for Children (WISC-IV) or the Wechsler Adult Intelligence Scale (WAIS-IV), depending on patient age. Full-scale IQ (FSIQ) was classified according to standard cut-off values: Normal cognitive function: FSIQ ≥ 85; Borderline cognitive function: FSIQ 70–84; Mild intellectual disability (ID): FSIQ 55–69M; Moderate ID: FSIQ 40–54; Severe ID: FSIQ < 40. For neuropsychiatric comorbidities, the presence of autism spectrum disorder (ASD) was determined using the Autism Diagnostic Observation Schedule-2 (ADOS-2) and Autism Diagnostic Interview-Revised (ADI-R), with classification based on DSM-5 criteria. Attention-deficit/hyperactivity disorder (ADHD) was diagnosed using the Conners Rating Scale and DSM-5 clinical criteria. Behavioral disorders, including oppositional defiant disorder (ODD) and anxiety disorders, were assessed using the Child Behavior Checklist (CBCL) and clinical interviews. For sleep disorders, a structured sleep history was taken, and the Pittsburgh Sleep Quality Index (PSQI) and Sleep Disturbance Scale for Children (SDSC) were used to classify sleep disturbances. Specific Learning Difficulties (SLD), including dyslexia, dyscalculia, and dysgraphia, were assessed using standardized academic achievement tests appropriate for age and national guidelines. The following assessments were employed: Reading and dyslexia : Batteria per la Dislessia e Disortografia Evolutiva (DDE-2) for Italian-speaking children. Mathematics and dyscalculia : Test AC-MT (Test di Abilità di Calcolo e Matematica) for evaluating mathematical abilities. Writing and dysgraphia : BVSCO-2 (Batteria per la Valutazione della Scrittura e della Competenza Ortografica) for assessing handwriting and spelling difficulties. All assessments were conducted at specialized centers with expertise in TSC, ensuring standardized evaluation across the cohort. Statistical Analysis Quantitative data were analyzed using Student’s t-test, while categorical variables were assessed using the Chi-square test. Statistical significance was determined for variables including age, onset of epilepsy, and frequencies of neurological manifestations. Percentages and proportions were calculated for categorical data, with significance assessed through a 2x2 contingency table analysis. RESULTS TSC1 Patients The TSC1 group included 31 patients (15 men, 16 women), with an average age of 25.1 years (range: 2–60 years). Of these, 45.1% had inherited mutations, while 54.9% were sporadic. TSC1 mutations were widely distributed along all the gene, involving in particular exons 4, 5, 6, 10, 15, 17, 18, 21 and 22. One patient presented an intronic variant (intron 1). Mutations were intragenic deletions (48.4%), nonsense (38.7%), missense (9.6%), and in one case a duplication (3.2%) (supplementary table 1 ). Among these patients, six came from the same family and were affected by the already reported c.1498C > T, p.(Arg500Ter) mutation (Fig. 1 a). TSC1 patients were affected by different types of seizures, differing in EEG findings, clinical course, age of onset and type of seizures. Epileptic seizures were present in 64.5% of patients, with onset averaging at 35.4 months. Seizure types were both generalized and focal crisis and some patients experienced multiple seizure types throughout life. More in detail, two patients (6.4%) presented infantile spasms, 12 (38.7%) had focal crises (3 with impaired awareness), 6 (19.3%) patients experienced generalized tonic clonic seizures, 4 (12.9%) suffered absence, one (3.2%) presented gelastic seziures, one (3.2%) reported seizures with fever, three (9.6%) suffered hypertonic crisis and one (3.2%) experienced atonic crisis. Seizure frequency ranged from 1 per lifetime to 28 per week (mean: 10.4 weeks, SD: 9.2). Severity and duration of crisis varied among patients and in the same patient. EEG abnormalities included spikes, waves, and hypsarrhythmia. Particularly, 11 patients (35.5%) had normal EEG, 1 (3.2%) presented hypsarrhythmia, 13 (41.9%) were reported to have spikes and waves (unilateral or bilateral), 11 (35.5%) had synchronous or asynchronous focal spikes, 1 (3.2%) had “arceau-like” pattern, 4 (12.9%) presented theta waves pattern. Epilepsy course varied from spontaneous remission to drug resistance, with valproic acid and carbamazepine being the most common treatments: 10 out of 20 patients (50%) achieved remission (complete response), while one (5%) had good response, 2 (10%) partial response, and the other and other 7 (35%) showed a drug-resistant epilepsy. Cognitive impairment levels of TSC1 patients ranged from mild to severe: 51.6% had normal cognitive functions, 9.7% had cognitive function near lower limit (borderline), 19.3% presented mild intellectual disability, 3.2% suffered from moderate intellectual disability and 16.1% had severe cognitive deficit. Autism spectrum disorder (ASD) was observed in four patients (12.9%), with only one patient showing Level 2 autism. Behavioral disorders (e.g., ADHD, anxious depressive syndrome, borderline personality) affected 8 patients (25.8%), while 12 (38.7%) experienced learning disabilities. Sleep disorders were reported in only 9.7%. Brain abnormalities of TSC1 group included cortical tubers (67.8% with > 5 tubers, mean size 8 mm) and subependymal nodules (51.6% with > 5 SENs, variable in size). Subependymal giant cell astrocytomas (SEGAs) were rare, found in only 2 patients (6.5%) probably related to a more severe genotype (non-sense mutations). White matter abnormalies and cerebral cysts were uncommon in our group of TSC1 patients, only 1 patient (3.2%) carrying the nonsense mutation c.2293C > T, had many cerebral cysts. Some mutations were associated with specific clinical patterns. The mutation c.1888_1891delAAAG (found in two patients) was associated in both patients with many bilateral tubers and SENs, while epilepsy and neuropsychiatric disorders were heterogeneous, suggesting a potential role of other modifying factors. Mutations c.1004delC and c.2111_2112delAT were linked to minimal neurological symptoms and a good quality of life, indicating a milder impact. The c.1498T mutation (associated to a common specific EEG pattern and many brain hamartomas) was found in 6 patients of the same family (Fig. 1 a) and five of them showed the same EEG pattern (sharp theta waves and spikes in the left temporal lobe or bilateral). Notably, in this family, not all the patient showed epilepsy, and some of those affected were partial responders to drugs, while other drug-resistant. Cognitive functions and behavioral disorders were variable, with different grade of intellectual disability (absent to moderate/severe) TSC2 Patients This study analyzed 50 patients with TSC2 gene mutations, comprising 21 males (42%) and 29 females (58%), with a mean age of 27.1 years (SD = 18.6, range 2–71 years). Mutations were classified as familial (23 cases, 46%) or de novo (27 cases, 54%) and showed extensive distribution across exons and included deletion (60%), nonsense (26%), missense (10%), and splicing mutations (6%). Furthermore, 4 intronic deletions were detected in this group (one spanning from intron 1 to exon 8), as well as a somatic mosaicism c.1373C > T (p.Arg1200Trp), involving 11.4% of the patient’s genome. Among these patients, two large families were included: one (5 members) harbored the c.1096G > T; (p.Glu366*) mutation (Fig. 1 b). The other one comprised 16 members (among the 50 known members of this family), and presented the already reported c.3693_3696delGTCT (p.Ser1232Thrfs*92) mutation (Fig. 1 c). Of the 50 patients, 36 (72%) experienced seizures, with a mean onset age of 29.45 months (SD = 66.3, range one month to 23 years). Seizure types varied, with 32% experiencing infantile spasms, 30% focal seizures (16% with impaired awareness) and 22% generalized tonic-clonic seizures. The frequency and severity of seizures ranged widely, with patients experiencing between 2 lifetime episodes to as many as 42 episodes per week (mean: 18.3, SD: 24.5). EEG patterns were varied, including theta waves, spikes, hypsarrhythmia, and focal spikes. Eighteen patients (36%) had normal EEG results, while focal and generalized spikes were more frequently observed. EEG patterns fluctuated over time within individual patients. The clinical course of epilepsy in TSC2 group of patients was variable, going from remission to multidrug resistance. Different antiseizures medications were used, including valproic acid, vigabatrin, carbamazepine, ACTH, topiramate, phenobarbital, levetiracetam, oxcarbazepine, lamotrigine, everolimus. Among them the most used were valproic acid and carbamazepine. TSC2 patients had been divided according to drug response into complete responder, (13 out of 36 patients- 36.1%), good responders (4 patients, 11%), partial responders (6 patients, 16.6%) and non-responders (9 patients, 25%). Cognitive impairment was prevalent among TSC2 patients, with 24%, 10%, and 24% exhibiting mild, moderate, and severe intellectual disability, respectively, while 42% displaying normal cognitive function. ASD was diagnosed in 12% of patients, while 48% had learning disabilities. Behavioral issues were present in 30% of patients, predominantly ADHD (20%), irritability (10%), and oppositional behavior (10%). It is necessary to underline that many patients presented more than one behavior disorder. Sleep disorders were rare, with only a few cases of insomnia or enuresis reported. MRI analyses revealed that 10% of TSC2 patients had no cortical tubers, while 74% had multiple tubers (> 5). The average tuber sizes was 17 mm (SD = 18.3mm), with some patients presenting cerebellar or cystic tubers. Subependymal nodules (SENs) were identified in 86% of patients, with dimensions varying widely (average size = 9.4mm, SD = 0.9mm). SEGAs were less common, found in 14% of patients. The average size of SEGAs was 1.3cm (SD = 15mm), ranging from 1cm to 1.5cm. In our cohort SEGAs were associated with different genotypes, in particular the mutations c.3094C > T, c.5201_5216dupATATCTACCCCTCCAA and c.1238_1285delCCT. One patient harboring 2 SEGAs carried a large intragenic deletion (spanning from exon 3 to 9), that could explain the increased number of SEGAs and a more severe neuropsychiatric and epileptic phenotype. Radial bands were noted in 12% of cases, mostly in parietal and temporal regions. Most patients (74%) had no white matter abnormalities, the remaining cases ranged from multiple to unspecified abnormalities. Only 3 patients (6%) had brain cysts and 2 (4%) had atrophy or hypoplasia of the corpus callosum. Regarding the specific mutations c.3693_3696delGTCT, (p.Ser1232Thrfs*92), it was found in one of the biggest family of TSC patients in Italy. The family is made up of at least 43 members (Fig. 1 c), of whom at least 25 members are affected by tuberous sclerosis. Complete information about genotype and phenotype of all the members was impossible to collect for many reasons (for example, some of them died without certain diagnosis, others refused clinical and genetic investigations, data were stored in different hospitals): for this reason, a total of 16 family members have been included in this study. Although they all carry the same mutation, an intrafamilial variable clinical presentation was observed, going from mild cutaneous phenotype to severe mental retardation and drug-resistant epilepsy. Nine patients from this family presented epilepsy. The median age of onset of epilepsy was 6 years old, ranging from birth to 23 years. Types of seizures and frequency were variable: infantile spasms, tonic clonic, myoclonic, focal, febrile crisis are only few examples. The most frequent were infantile spasms, tonic clonic and focal crises with impaired awareness. Regarding the EEG, the most frequent patterns were hypsarrhythmia and focal spikes (with secondary generalization) in the left frontal temporal lobes: it must be underlined that all the normal EEGs found in TSC2 epileptic patients were observed in this family. Drug response was different among the members of this family, and the biggest group is made up of good/partial responders, although there were 4 cases of remission. All the levels of severity of cognitive impairment were present in this family, although the most frequent was mild intellectual disability. ASD was rare in this family and none of them reached the diagnostic criteria for confirmed diagnosis, only some of them had a sub-clinical form of ASD with limited social difficulties. Learning disability was present mainly in patients with moderate-severe intellectual disability, who were the minority of the family. The most frequent behavior disorders were ADHD and oppositional disorders. Sleep disorders were basically absent. Many bilateral tubers and SENs were reported in all the members of the family, while none of them presented SEGAs. Cerebral cysts and white matter abnormalities were absent, while some of them presented radial bands. To sum up the common characteristics of this family members were multiple tubers and SENs, absence of SEGAs, attention deficit disorder, mild mental retardation (that could explain why they managed to reproduce), partial epilepsy control, infantile spasms and partial complex crisis. The mutation c.1096G > T (p.Glu366Ter) was present in another family (patients 69,70,71,72,73) (respectively a father and his two sons and two daughters). The types of seizures described in this family were focal with impaired awareness and infantile spasms. All patients also shared severe intellectual and learning disability. ASD, behavior and sleep disorders were absent. Brain MRI reports revealed the presence of few to many tubers and SEN, but no-one showed SEGA. TSC1 vs. TSC2 Patients The study cohort included 81 patients, divided into TSC1 and TSC2 groups. Tables 1 – 3 summarize the comparison between the two groups and the statistical data. Table 1 Main Clinical Characteristics of the two groups of patients TSC1 patients TSC2 patients p value Number of Patients 31(38,3%) 50(61.7%) - Females 16(51.6%) 29(58%) NS Average Age (Standard Dev) [Years] 25.1(16.9) 27.1(18.6) NS Age Range [Years] 2–60 2–71 NS De Novo Mutations 18(58.1%) 27(54%) NS Familial Mutations 13(41.9%) 23 (46%) NS Intragenic Deletions 15(48.4%) 30(60%) NS Intragenic Duplications 1(3.2%) 2(3.3%) NS Missense Mutations 3(9.6%) 5(10%) NS Nonsense Mutations 12(38.7%) 13(26%) NS Table 2 Epilepsy and Neuropsychiatric comorbidities of the two groups of patients (included if at least 5 individuals presented the specific feature) * Patient 9, 44 and 49 - experiencing only one to two seizures in their lifetime – are excluded from this range. TSC1 patients TSC2 patients p value Seizures Mean Age at epilepsy onset (SD) [Months] 35.4(25.8) 29.45(66.3) < 0.01 Age range of epilepsy onset [Months] 1–96 1-276 NS Patients Without Seizures 11(35.5%) 14(28%) NS Infantile Spasms 2(6.4%) 16(32%) 0.016 Focal seizures 12(38.7%) 15(30%) NS Focal seizures with impaired awareness 3(9.6%) 8(16%) NS Generalized tonic-clonic seizures 6(19.3%) 11(22%) NS Absence Seizures 4(12.9%) 4(8%) NS Hypertonic seizures 3(9.6%) 5(10%) NS Seizure Frequency (SD) [episodes per week] 10.4 (9.1) 18.3 (24.5) NS (0.089) Range of seizures per week 1–28* 1–42* NS Remission of Seizures [> 75% of reduction] 10(50%) 13(36.1%) NS Partial/ good response (25 to 74.9% of reduction) 3(15%) 10(27.6%) NS Drug Resistant patients 7(35%) 9(25%) NS EEG findings Normal 11(35.5%) 18 (36%) NS Hypsarrhythmia 1(3.2%) 5(10%) NS Bilateral Spikes and Waves Pattern 7(22.6%) 12(24%) NS Focal Anomalies 8(25.8%) 15(30%) NS Theta Waves Pattern 4(12.9%) 6(12%) NS Neuropsychiatric comorbidities Normal Cognitive Functions 16(51.6%) 21(42%) NS Mild Intellectual disability 6(19.3%) 12(24%) NS Moderate Intellectual disability 1(3.2%) 5(10%) NS Severe Intellectual disability 5(16.1%) 12(24%) NS Patients Without Autism Spectrum Disorder 27(87.1%) 44(88%) NS Autism Level 1 and 2 4(12.9%) 6(12%) NS No learning Disability 19(61.3%) 26(52%) NS Learning Disability 12(38.7%) 24(48%) NS Absence of Behavior Disorders 23(74.2%) 35(70%) NS ADHD 3(9.7%) 10(20%) NS Irritability And Agitation 3(9.7%) 5(10%) NS Oppositional Disorder/Aggression 0 5(10%) NS Absence of Sleep disorders 28(90%) 48(96%) NS Table 3 MRI finding of the two groups of patients. SD: Standard Deviation; SEGA: Subependymal giant cell astrocytoma. TSC1 patients TSC2 patients p value Tubers Absence of tubers 5 (16%) 5 (10%) NS 1 to 5 tubers 5 (16.1%) 7 (14%) NS More than 5 tubers 21 (67.8%) 36 (72%) NS Mean Size Of Tubers (SD) 8 mm (3.9) 17mm (18.3) < 0.01 Tubers Dimensions range (95% C.I.) 5–20 mm 4–30 mm NS Subependymal nodules Absence of Subependymal nodules 5(16.1%) 11(22%) NS 1 to 5 Subependymal nodules 10(32.3%) 2(4%) NS More than 5 subependymal nodules 16(51.6%) 31(62%) NS Average size of subependymal nodules (SD) 3.87 mm (2.09) 9.4 mm (0.9) < 0.01 Subependymal nodules dimensions range (95% C.I.) 2-10mm 5–12 mm NS Subependymal giant-cell astrocytomas Patients without SEGA 29 (93.6%) 43 (86%) NS 1 SEGA 2 (6.4%) 6 (12%) NS 2 or more SEGA 0 1 (2%) NS Patients underwent SEGA surgery 1 (50%) 4 (57%) NS Average size Of SEGAs (SD) 1.1 cm (0.2) 1.3 cm (0.15) NS SEGA dimensions range (95% C.I.) 1 -1.2cm 1-1.5 cm NS Radial Bands Presence of Radial Bands 12 (38.7%) 6 (12%) 0.011 1 Radial Band 3 (9.7%) 2 (4%) NS 2 or more Radial Bands 9 (29%) 4 (8%) 0.028 Cerebral Cysts Absence of Cerebral Cysts 30 (96.7%) 47 (94%) NS 1 Cerebral Cyst 0 2 (4%) NS 2 or more Cerebral Cysts 1 (3.2%) 1 (2%) NS White Matter Abnormalities Absence of White Matter Abnormalities 22 (71%) 37 (74%) NS Limited White Matter Abnormalities 4 (12.9%) 5 (10%) NS Diffuse White Matter Abnormalities 5 (16.1%) 8 (16%) NS The proportion of TSC2 patients (61.7%) was higher than TSC1 ones (38.3%). There was not statistically significant difference between TSC1 and TSC2 groups with respect to sex ( TSC1 : male 48.4%-female 51.6%; TSC2 : male 42%-female 58%) and to the mean age of patients. In our cohort de novo and familial mutations were proportionally distributed between the two groups. There was no statistically significant difference for types of mutations, which included intragenic deletions, duplications, missense and nonsense mutations. TSC2 patients exhibited a high variability in clinical manifestations, seizure types, cognitive outcomes, and behavioral disorders. TSC2 group had a higher prevalence of epilepsy than TSC1 patients (72% vs. 64.5%, not statistically significant) and in particular of infantile spasms (p = 0.016). About other type of seizures there was not any significant difference between the two groups: focal crisis (p = 0.3651), tonic clonic crisis (p = 0.94), gelastic crisis (p = 0.72), focal dyscognitive crisis (p = 0.41), febrile crises (p = 0.38), atonic crisis (p = 0.87), hypertonic crisis (p = 0.96), epileptic status (p = 0.25) and myoclonic crisis (p = 0.16). There was no statistically significant difference in the number of weekly episodes between the two groups, but a tendency of TSC2 patients to present more seizure per week (p = 0.089). Regarding EEG, a normal pattern was found in the same proportion of TSC1 and TSC2 patients. The only appreciable difference between TSC1 and TSC2 patients was in the prevalence of hypsarrhythmia (higher in TSC2 patients, even if not statistically significant). Both groups showed similar responses to antiseizure medications, with no significant difference in rates of remission, partial or good response, or drug-resistance. Cognitive and behavioral disorders were similarly distributed, although TSC1 patients had higher rates of borderline cognitive function, while TSC2 patients had more severe neuropsychiatric profiles compared to TSC1 (not statistically significant). Importantly, patients presenting remission from seizures were likely to have normal cognitive profile: among the TSC1 responders, 70% did not show any intellectual disability (90% considering also those mildly affected). Among TSC2 patients, 28.6% showing remission did not show any intellectual disability, and the ration increased to 78.6% if considering normal to mild intellectual disability. Tubers, SENs, and radial bands occurred at comparable frequencies, with TSC2 patients slightly more likely to have multiple and larger tubers (p < 0.01) and larger SEN (p < 0.01), while TSC1 patients to have radial (p < 0.05) and multiple bands (p < 0.01). DISCUSSION This study aimed to conduct a mutational screening of a cohort of Sicilian TSC patients, compare the clinical features of TSC1 and TSC2 groups, and described four new potential genotype-phenotype correlations. The cohort includes 81 patients, representing approximately 15% of the estimated TSC population in Sicily, assuming a prevalence of 1 in 8,000 individuals. This proportion suggests that our study provides a significant and representative sample, offering valuable insights into the genetic and phenotypic spectrum of the disease in this region. Given the focus on Sicily, it is relevant to underline that, despite the application of advanced molecular techniques, certain challenges may limit the full identification of TSC cases, including potential underdiagnosis due to disparities in healthcare access and variability in disease expression: patients with milder phenotypes can remain undiagnosed, particularly in cases where dermatological or renal manifestations are subtle, and epilepsy is absent or well-controlled. However, genetic testing and specialized care are becoming increasingly accessible across all Sicilian provinces, enhancing the identification and diagnosis of TSC cases. These factors highlight the need for continued efforts in optimizing TSC surveillance and diagnostic strategies in this population. The proportion of TSC2 patients was higher than TSC1 , consistent with literature indicating that approximately two-thirds of pathogenic variants are TSC2 , and one-third are TSC1 [ 13 ]. Gender distribution and mean age were similar between the two groups, in agreement with previous studies [ 14 , 15 ]. De novo mutations represented 54.3% of the total, while familial mutations accounted for 45.6%. This differs from literature suggesting a greater disparity (approximately 2:1 de novo vs familial cases), likely due to challenges in accurately reconstructing family histories in our cohort [ 16 , 17 ]. There was no significant difference in the proportions of de novo and familial mutations between the TSC1 and TSC2 groups (p = 0.9413), differently from literature data that suggest a higher proportion of TSC2 mutations in sporadic cases [ 14 , 18 – 20 ]. The type of mutations – deletions, duplications, missense, and nonsense – were equally distributed, diverging from studies that describe distinct distributions for TSC1 (predominantly nonsense and deletions) and TSC2 (mainly frameshift, nonsense, and deletions) [ 15 , 21 , 22 ]. This discrepancy may reflect our smaller sample size. However, some reports align with our findings, describing similar proportions of missense and other type of mutations in TSC2 patients [ 20 ]. Consistent with previous studies, TSC1 patients had a lower probability of experiencing seizures compared to TSC2 patients, even without statistical significance (35.5% of TSC1 patients did not present seizures, vs. 28% of TSC2 patients) [ 14 ]. However, these last showed a significantly higher prevalence of infantile spasms (p = 0.016), a finding aligned with earlier studies (19% in TSC1 vs. 41% in TSC2 ) [ 19 – 21 ]. Interestingly, the age of epilepsy onset was similar (but patients with TSC2 mutations tended to exhibit a higher number of seizures per week), partially in line with literature that suggests an earlier onset and more frequent drug-resistant epilepsy in TSC2 [ 21 – 24 , 31 ]. This could be due to limitations in seizure quantification or the dynamic nature of drug responsiveness. About drug responsiveness, percentages of seizure remission, good or partial responders and drug-resistant patients were comparable in TSC1 and TSC2 group, even if TSC1 patients tend to show a higher ratio of seizures remission (50% vs. 36.1%), but a lower amount of partial or good responders (i.e. seizure reduction from 25 to 74.9%), which was higher in TSC2 patients (27.6% vs 15%). These results are somewhat in contrast with other studies, that clearly affirm that TSC2 patients suffer more severe and drug-resistant epilepsy [ 24 – 27 ]. In the same way, EEG findings were substantially similar in the two groups, with a higher proportion of TSC2 patients showing hypsarrhythmia, even if not statistically different. We found no significant difference in the severity of intellectual disability between TSC1 and TSC2 patients, although mild intellectual disability was more prevalent in TSC1 patients (p = 0.225). This partially contrasts with reports associating more frequently TSC2 individuals with severe intellectual impairment, likely influenced by epilepsy severity and tuber burden (20% TSC1–51 % TSC2 ) [ 28 – 31 ]. Interestingly, specific TSC1 and TSC2 mutations have been linked to both mild and severe phenotypes, illustrating the heterogeneity within and between genotypes. Nevertheless, the association between TSC2 mutations and severe intellectual disability is not constantly present. Some TSC2 genotypes have been found to be associated with mild phenotype without cognitive impairment, such as c.1514G > A (p.Arg505Gln), c.3598C > T (p. Arg1200Trp) and c.2714G > A (Arg905Gln). On the other hand, some specific TSC1 mutations have been associated with intellectual disability such as c.682C > T (p.Arg228Ter), c.1117C > T (p.Gln373Ter), c.2713C > T (p.Arg905Trp) and c.2713C > G (p.Arg905Gly) [ 33 – 40 ]. Furthermore, a different severity of intelligence deficit has been described in the same mutational group: for example, patients with a TSC1 variant in the tuberin interacting domain showed significantly lower IQ compared to other TSC1 variants. Similarly, mutations causing truncation of the proximal part of TSC2 or involving the hamartin-interacting domain cause more severe intellectual disability than small in-frame deletions in the distal part of the gene or missense mutations. These findings provide further evidence that different types and locations of TSC1 or TSC2 pathogenic variants may be associated with distinct neurocognitive phenotypes [ 24 , 27 , 31 , 40 , 41 ]. Importantly, patients who experienced seizure remission were more likely to have a normal cognitive profile. Among TSC1 responders, 70% had no intellectual disability, and this proportion rose to 90% when including those with mild intellectual impairment. In the TSC2 group, 28.6% of patients with seizure remission had no intellectual disability, increasing to 78.6% when considering both normal cognitive profiles and mild intellectual disabilities. On the other hand, among the drug-resistant patients, intellectual disability was severe in 4 out 7 TSC1 drug-resistant patients (57.1%) and in 6 out of 9 (66.6%) TSC2 drug-unresponsive patients (see supplementary table 1 ). These results are consistent with the literature and support the current view that preventing the development of drug resistance may help preserve or improve cognitive outcomes in patients with TSC [ 41 – 44 ]. No significant differences emerged in the prevalence of learning disabilities, ASD, or behavioral disorders, including ADHD, irritability, borderline personality, and sleep disturbances. These findings contrast with reports of higher ASD prevalence and behavioral challenges in TSC2 patients [ 44 ]. The lack of significant differences may reflect the small cohort size of patients presenting these neuropsychiatric conditions. Tuber prevalence was similar between groups, though TSC1 patients were more likely to have smaller tubers (p < 0.01). This aligns with some studies but contrasts with reports of a higher cortical tuber burden in TSC2 (91% vs 76%) [ 13 , 21 , 43 ]. SENs, SEGAs, and white matter abnormalities were equally frequent, though TSC1 patients exhibited a higher presence of radial bands (p = 0.011) and a more likely occurrence of more than two bands (p = 0.028): this represents a previously unreported finding in patients with TSC. The main reviews published so far report that TSC2 patients usually present a more severe phenotype, for nearly all the features. However, there can be overlap in disease severity between the two groups, indeed many TSC1 patients were found to have severe neurocutaneous phenotype and some patients with TSC2 mutations had mild phenotype, representing exceptions to the general rule [ 20 – 22 , 29 , 31 , 43 ]. Therefore, in our opinion, clinicians should not strictly rely on genotype for clinical counseling and prognosis, unless a specific genotype-phenotype correlation is available. In this regard, genotype-phenotype correlations may be challenging in clinical practice, due to the high inter- and intra-familial phenotype variability, the age dependence onset of certain manifestations, the role of genetic and environmental modifiers, including cases of mosaicism, and the rare occurrence of specific (unknown) variants. Despite that, genotype–phenotype correlation is essential for clinical management, prognosis and personalized treatments, and are currently used by the clinicians to oversee the outcome of TSC patients, especially in some specific cases. Based on the genetic and clinical data from this study, we explored four tentative genotype–phenotype associations in families with multiple affected individuals. However, given the marked intrafamilial phenotypic variability observed, these associations should not be interpreted as strict correlations, but rather as hypothesis-generating observations requiring further validation. The TSC2 mutation c.3693_3696delGTCT (p.Ser1232Thrfs*92) was found in a large family (43 members, 25 affected, 16 reported in the present study). The clinical presentation was variable, going from mild cutaneous phenotype to severe intellectual disability and drug-resistant epilepsy. The shared features included multiple tubers and SENs, ADHD, mild intellectual disability (which may explain the high reproductive rates in certain individuals), partial/good seizure control, infantile spasms and focal seizures with impaired awareness. Unfortunately, complete information about genotype and phenotype of all the members was impossible to collect for many reasons (some of them had died without certain diagnosis, others refused clinical and genetic investigations, some data were stored in different hospitals and clinics). The TSC2 mutation c.1096G > T (p.Glu366Ter) was observed in five-member family, associated with severe intellectual disability and specific seizure types (focal crises with impaired awareness and infantile spasms). The TSC1 mutation c.1498T (p.Arg500Ter) identified in six members of a large family, could be associated to a distinct EEG pattern (sharp theta waves and spikes in the left temporal lobe or bilateral) and multiple brain tubers and SENs, but no SEGAs. Despite that, cognitive functions were variable and not every patient presented seizures, but those affected were partial responders. The TSC1 mutation c.2111_2112delAT (p.Tyr704Ter) was found in three patients of a small family, with a mild neurological phenotype, multiple bilateral tubers and SENs but no seizures or neuropsychiatric disorders. In addition to the reported observations that (sometime) phenotypes associated with TSC1 and TSC2 pathogenic variants overlap substantially, we did not always observe significant differences in relation to the type of gene mutation in our retrospective analysis. However, overall results and genotype-phenotype correlations observed in this study reinforce evidence from larger cohorts, particularly those indicating that TSC2 mutations result in a more severe clinical phenotype, than TSC1 mutations. Our data reveal that TSC2 patients face a significantly higher burden of epilepsy and EEG abnormalities (in particular infantile spasms and hypsarrhythmia), supporting existing evidence that TSC2 mutations may drive hyperactivity within the mTOR pathway more forcefully than TSC1 , thereby increasing neural excitability [ 24 ]. Such findings underscore the importance of identifying TSC genotypes early, as TSC2 patients may benefit from proactive epilepsy management with vigabatrin, cannabidiol or mTOR-inhibitors [ 45 – 48 ]. The higher rate of EEG abnormalities in TSC2 patients, including hypsarrhythmia and focal spikes, correlates with findings by Chu-Shore et al., who reported similar EEG profiles in TSC2 patients with refractory epilepsy [ 49 ]. These EEG patterns, frequently associated with epileptic encephalopathies, suggest that tubers and associated neural dysplasia in TSC2 patients might extend beyond visible MRI lesions, potentially involving perituberal regions [ 50 ]. The neuropsychiatric profile of TSC2 patients, including a higher prevalence of ADHD and severe cognitive impairment, suggests that TSC2 mutations exert a greater impact on cognitive and behavioral domains, possibly through disruption of cortical networks involved in attention and executive function [ 31 , 32 ] Our data confirm findings from Bolton et al. and others who observed a correlation between TSC2 mutations and ASD severity, underscoring the need for early intervention strategies, particularly for TSC2 patients at risk of developmental delays and behavioral disturbances [ 29 – 32 , 44 ]. These findings highlight the value of genotype-specific approaches in managing TSC patients. Given the severe phenotype associated with TSC2 mutations, early mTOR inhibitor therapy may be beneficial, not only for seizure control but also potentially for reducing neuropsychiatric symptoms, as suggested by recent clinical trials [ 45 – 47 ]. Furthermore, given the correlation between TSC1 and radial band prevalence, also TSC1 patients might benefit from neurodevelopmental monitoring, even in the absence of severe epilepsy, to address potential subclinical impacts on cognitive function. While our findings align with existing literature, the limited cohort size necessitates caution when generalizing results. Further multicenter studies with larger sample sizes could provide a deeper understanding of rare features and refine genotype-specific treatment guidelines. Additionally, longitudinal studies are essential to evaluate the long-term impact of early genotype-informed interventions on neurodevelopmental and psychiatric outcomes in TSC patients. CONCLUSIONS This study represents the first comprehensive TSC mutational analysis and genotype-phenotype correlation conducted in Sicilian TSC patients. Our findings reinforce the established association of TSC2 mutations with more severe neurological manifestations, including infantile spasms and related EEG abnormalities. Conversely, patients harboring TSC1 mutations showed a higher prevalence of radial bands, and milder cognitive involvement, emphasizing the nuanced heterogeneity of TSC. The proposed genotype-phenotype correlations highlight the variability within TSC manifestations and underscore the need for personalized approaches to management. Our results support early genetic testing to guide treatment strategies, particularly for TSC2 patients who may benefit from mTOR-inhibitor therapies and proactive epilepsy management. Larger multicenter and longitudinal studies are essential to confirm these correlations, refine genotype-specific therapeutic guidelines, and evaluate long-term outcomes of early interventions. By leveraging genetic insights, clinicians can advance precision medicine in TSC, improving quality of life and clinical outcomes for affected patients. Declarations Ethics approval and consent to participate : Approvement of the Ethical committee of the University has been obtained, as well as consent to participate from patients and/or parents or legal guardians of minor patients. Consent for publication: Written informed consent for publication of this study was obtained from all adult patients and the parents or legal guardians of minor participants. Availability of data and material : The datasets generated and/or analyzed during the current study are available in the ClinVar repository (https://www.ncbi.nlm.nih.gov/clinvar/) and can be retrieved using the following submission IDs: 15233733; 15233796; 15233928; 15233938; 15233951; 15233966; 15233980; 15233928; 15234011; 15234019; 15234029; 15234041; 15234045; 15234142; 15234050; 15234063; 15233951; 15234070; 15234109 15234148; 15234153; 15234159; 15234185; 15234192; 15234203; 15237360; 15234242; 15234212; 15234220; 15234224; 15234375; 15234222; 15234237; 15234362; 15237397; 15234207; 15234240; 15237421, which will be linked to public variant accession numbers upon final processing. The data can be accessed upon reasonable request from the corresponding author. Conflict of Interest : The authors report no conflict of interest in the subject presented by the present research Funding : No funding has been received for the preparation of this article Authors’ contribution : ADP wrote the first draft of the manuscript; CD, ED and SS performed the literature research; MP, FC, RS, MV, AZ and MB followed-up the patients; ME and MLB validated the results; AP & MR critically revised the manuscript Acknowledgements : The Authors wish to thank the “Associazione Sclerosi Tuberosa” (AST) for its support; The Authors also wish to thank the Health Operational Plan (FSC 2014-2020) “Trajectory 4-Biotechnology, Bioinformatics and Pharmaceutical Development” entitled “PHARMA-HUB” References Osborne, J. P. et al. Epidemiology of tuberous sclerosis. Ann. N Y Acad. Sci. 615 , 125–127 (1991). Gómez, M. R. et al. History of the tuberous sclerosis complex. Brain Dev. 17 Suppl , 55–57 (1995). Brigo, F. et al. First descriptions of tuberous sclerosis by Désiré-Magloire Bourneville (1840–1909). Neuropathology 38 , 577–582 (2018). Huschner, F. et al. Molecular EPISTOP, a comprehensive multi-omic analysis of blood from Tuberous Sclerosis Complex infants age birth to two years. Nat. Commun. 14 (1), 7664 (2023). Kandt, R. S. et al. Linkage of an important gene locus for tuberous sclerosis to a chromosome 16 marker for polycystic kidney disease. Nat. Genet. 2 , 37–41 (1992). Tyburczy, M. E. et al. Mosaic and intronic mutations in TSC1/TSC2 explain the majority of TSC patients with no mutation identified by conventional testing. PLoS Genet. 11 , e1005637 (2015). Ye, Z. et al. Mosaicism in tuberous sclerosis complex: lowering the threshold for clinical reporting. Hum. Mutat. 43 , 1956–1969 (2022). Inoki, K. et al. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat. Cell. Biol. 4 , 648–657 (2002). Northrup, H. et al. Updated international tuberous sclerosis complex diagnostic criteria and surveillance and management recommendations. Pediatr. Neurol. 123 , 50–66 (2021). Curatolo, P. et al. Neurological and neuropsychiatric aspects of tuberous sclerosis complex. Lancet Neurol. 14 , 733–745 (2015). Pinto, A. L. R. et al. Neurological manifestations of tuberous sclerosis complex: the importance of early diagnosis. Arq. Neuropsiquiatr. 80 , 983–984 (2022). Nabbout, R. et al. Historical Patterns of Diagnosis, Treatments, and Outcome of Epilepsy Associated With Tuberous Sclerosis Complex: Results From TOSCA Registry. Front. Neurol. 12 , 697467 (2021). Dabora, S. L. et al. Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. Am. J. Hum. Genet. 68 , 64–80 (2001). Northrup, H., Koenig, M. K. & Pearson, D. A. Tuberous sclerosis complex. Tuberous Sclerosis Complex. Peron, A. et al. Genetics, genomics, and genotype-phenotype correlations of TSC: insights for clinical practice. Am. J. Med. Genet. C Semin Med. Genet. 178 , 281–290 (2018). Curatolo, P. et al. Tuberous sclerosis. Handb. Clin. Neurol. 111 , 323–331 (2013). Langkau, N. et al. TSC1 and TSC2 mutations in tuberous sclerosis, the associated phenotypes and a model to explain observed TSC1/TSC2 frequency ratios. Eur. J. Pediatr. 161 , 393–402 (2002). Sancak, O. et al. Mutational analysis of the TSC1 and TSC2 genes in a diagnostic setting: genotype–phenotype correlations and comparison of diagnostic DNA techniques in tuberous sclerosis complex. Eur. J. Hum. Genet. 13 , 731–741 (2005). Au, K. S. et al. Genotype/phenotype correlation in 325 individuals referred for a diagnosis of tuberous sclerosis complex in the United States. Genet. Med. 9 , 88–100 (2007). Jones, A. C. et al. Comprehensive mutation analysis of TSC1 and TSC2-and phenotypic correlations in 150 families with tuberous sclerosis. Am. J. Hum. Genet. 64 , 1305–1315 (1999). Rosset, C. et al. TSC1 and TSC2 gene mutations and their implications for treatment in tuberous sclerosis complex: a review. Genet. Mol. Biol. 40 , 69–79 (2017). Kothare, S. V. et al. Severity of manifestations in tuberous sclerosis complex in relation to genotype. Epilepsia 55 , 1025–1029 (2014). Di Napoli, C. et al. TSC1 and TSC2: tuberous sclerosis complex and its related epilepsy phenotype. J. Pediatr. Neurol. 10.1055/s-0041-1727142 (2021). Zeng, L-H. et al. Tsc2 gene inactivation causes a more severe epilepsy phenotype than Tsc1 inactivation in a mouse model of tuberous sclerosis complex. Hum. Mol. Genet. 20 , 445–454 (2011). Nabbout, R., Kuchenbuch, M., Chiron, C. & Curatolo, P. Pharmacotherapy for Seizures in Tuberous Sclerosis Complex. CNS Drugs . 35 (9), 965–983 (2021). Nabavi Nouri, M. et al. Epilepsy management in tuberous sclerosis complex: existing and evolving therapies and future considerations. Pediatr. Neurol. 126 , 11–19 (2022). Henske, E. P. et al. Tuberous sclerosis complex. Nat. Rev. Dis. Primers . 2 , 16035 (2016). Feliciano, D. M. et al. The neurodevelopmental pathogenesis of tuberous sclerosis complex (TSC). Front. Neuroanat. ; 14 . (2020). Bolton, P. F. et al. Intellectual abilities in tuberous sclerosis complex: risk factors and correlates from the Tuberous Sclerosis 2000 Study. Psychol. Med. 45 , 2321–2331 (2015). Joinson, C. et al. Learning disability and epilepsy in an epidemiological sample of individuals with tuberous sclerosis complex. Psychol. Med. 33 , 335–344 (2003). de Vries, P. J. et al. Tuberous sclerosis complex-associated neuropsychiatric disorders (TAND): new findings on age, sex, and genotype in relation to intellectual phenotype. Front. Neurol. 11 , 603 (2020). van Eeghen, A. M. et al. Genotype and cognitive phenotype of patients with tuberous sclerosis complex. Eur. J. Hum. Genet. 20 , 510–515 (2012). Ekong, R. et al. Variants within TSC2 exons 25 and 31 are very unlikely to cause clinically diagnosable tuberous sclerosis. Hum. Mutat. 37 (4), 364–370 (2016). Farach, L. S. et al. TSC2 c.1864C > T variant associated with mild cases of tuberous sclerosis complex. Am. J. Med. Genet. A . 173 (3), 771–775 (2017). Jansen, A. C. et al. Unusually mild tuberous sclerosis phenotype is associated with TSC2 R905Q mutation. Ann. Neurol. 60 (5), 528–539 (2006). van Eeghen, A. M. et al. Central TSC2 missense mutations are associated with a reduced risk of infantile spasms. Epilepsy Res. 103 (1), 83–87 (2013). O’Connor, S. E. et al. A family with seizures and minor features of tuberous sclerosis and a novel TSC2 mutation. Neurology 61 (3), 409–412 (2003). Wentink, M. et al. Functional characterization of the TSC2 c.3598C > T (p.R1200W) missense mutation that co-segregates with tuberous sclerosis complex in mildly affected kindreds. Clin. Genet. 81 (5), 453–461 (2012). Khare, L. et al. A novel missense mutation in the GTPase activating protein homology region of TSC2 in two large families with tuberous sclerosis complex. J. Med. Genet. 38 (5), 347–349 (2001). Camposano, S. et al. Distinct clinical characteristics of tuberous sclerosis complex patients with no mutation identified. Ann. Hum. Genet. 73 (2), 141–146 (2009). Farach, L. S. et al. Drug-Resistant Epilepsy in Tuberous Sclerosis Complex Is Associated With TSC2 Genotype: More Findings From the Preventing Epilepsy Using Vigatrin (PREVeNT) Trial. Pediatr. Neurol. 159 , 62–71 (2024). De Ridder, J. et al. Early epileptiform EEG activity in infants with tuberous sclerosis complex predicts epilepsy and neurodevelopmental outcomes. Epilepsia 62 , 1208–1219 (2021). Curatolo, P. et al. Advances in the genetics and neuropathology of tuberous sclerosis complex: edging closer to targeted therapy. Lancet Neurol. 21 , 843–856 (2022). Moavero, R. et al. Early clinical predictors of autism spectrum disorder in infants with tuberous sclerosis complex: results from the EPISTOP study. J. Clin. Med. 8 (6), 788 (2019). Krueger, D. A. et al. Everolimus for treatment of tuberous sclerosis complex-associated neuropsychiatric disorders. Ann. Clin. Transl Neurol. 4 (12), 877–887 (2017). Hwang, S-K. et al. Everolimus improves neuropsychiatric symptoms in a patient with tuberous sclerosis carrying a novel TSC2 mutation. Mol. Brain . 9 , 56 (2016). Maász, A. et al. Three-Year Follow-Up after Intrauterine mTOR Inhibitor Administration for Fetus with TSC-Associated Rhabdomyoma. Int. J. Mol. Sci. 24 (16), 12886 (2023). Samanta, D. Evolving treatment strategies for early-life seizures in Tuberous Sclerosis Complex: A review and treatment algorithm. Epilepsy Behav. 161 , 110123 (2024). Chu-Shore, C. J., Major, P., Camposano, S., Muzykewicz, D. & Thiele, E. A. The natural history of epilepsy in tuberous sclerosis complex. Epilepsia 51 (7), 1236–1241 (2010). Coban, G. et al. Synthetic MRI in children with tuberous sclerosis complex. Insights Imaging . 13 (1), 115 (2022). Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1.xlsx SupplementaryTable2.xlsx Cite Share Download PDF Status: Published Journal Publication published 27 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 24 Apr, 2025 Reviews received at journal 22 Apr, 2025 Reviews received at journal 15 Apr, 2025 Reviewers agreed at journal 12 Apr, 2025 Reviewers agreed at journal 11 Apr, 2025 Reviewers invited by journal 10 Apr, 2025 Submission checks completed at journal 10 Apr, 2025 First submitted to journal 27 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5804009","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":441192142,"identity":"ee8a102e-548e-4033-b258-b9f3b86b4062","order_by":0,"name":"Andrea Domenico Pratico'","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYHACAzjrAAODDQMDMxuYw0OEFmaQljSEFjx6EFqA4DAQQ7TgtIZ/dvO2Dx9qGPLM288fPPCj4nzidna2xMcVFQwy9ji0SNw5VjxzxjGGYpkzyQwHe87cTtzZzHbY8MwZPA67kWPMzMPGkDiDIZnhMGPb7cQNh9nbJBvbcGuRB2n58w+ohf8xSMs5qJZ/uLUYgLQwtgG1SIBtOQDUwnZMsrEBtxbDG2nFjL19EsUSEo8NgH5JNgZqSTZsOCbBw3MAuxa5G8mbGX58s8mT4E98/OFHhZ3shvPHDB821NjYszfg8j8YSCRgiOBVDwIYWkbBKBgFo2AUwAEAZ1ZXKotNKz0AAAAASUVORK5CYII=","orcid":"","institution":"University Kore of Enna","correspondingAuthor":true,"prefix":"","firstName":"Andrea","middleName":"Domenico","lastName":"Pratico'","suffix":""},{"id":441192143,"identity":"a6403e9e-1337-4c68-8f7d-29af68a3068e","order_by":1,"name":"Claudia Di Napoli","email":"","orcid":"","institution":"University Kore of Enna","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"Di","lastName":"Napoli","suffix":""},{"id":441192144,"identity":"51166cc3-7cd0-4065-a9a2-11ca1448e96f","order_by":2,"name":"Stefania Salafia","email":"","orcid":"","institution":"Lentini Hospital","correspondingAuthor":false,"prefix":"","firstName":"Stefania","middleName":"","lastName":"Salafia","suffix":""},{"id":441192145,"identity":"6ffa2b8c-786d-4c00-a22b-0235c4327b00","order_by":3,"name":"Edoardo Dammino","email":"","orcid":"","institution":"University of Catania","correspondingAuthor":false,"prefix":"","firstName":"Edoardo","middleName":"","lastName":"Dammino","suffix":""},{"id":441192146,"identity":"f6b0f9fa-a659-4804-91fe-a349a90b1642","order_by":4,"name":"Maria Piccione","email":"","orcid":"","institution":"University of Palermo","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Piccione","suffix":""},{"id":441192147,"identity":"3ff27730-c85e-430d-a271-b43b63f2a1c5","order_by":5,"name":"Francesco Calì","email":"","orcid":"","institution":"Oasi Research Institute‐IRCCS","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"","lastName":"Calì","suffix":""},{"id":441192148,"identity":"a0eaa7cb-6658-4da0-b479-e7c646d4473f","order_by":6,"name":"Renato Scifo","email":"","orcid":"","institution":"Santa Marta and Santa Venera Hospital","correspondingAuthor":false,"prefix":"","firstName":"Renato","middleName":"","lastName":"Scifo","suffix":""},{"id":441192149,"identity":"fc2248cb-578e-4a39-83dd-ecc84df17703","order_by":7,"name":"Michele Vecchio","email":"","orcid":"","institution":"University of Catania","correspondingAuthor":false,"prefix":"","firstName":"Michele","middleName":"","lastName":"Vecchio","suffix":""},{"id":441192150,"identity":"c8124610-c32a-4de8-b36c-17de2224ca3f","order_by":8,"name":"Andrea Zonta","email":"","orcid":"","institution":"Azienda Ospedaliera Citta' della Salute e della Scienza di Torino","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Zonta","suffix":""},{"id":441192151,"identity":"c1c8f469-5343-40ca-a26d-9c3d3d2c3f80","order_by":9,"name":"Maria Bonsignore","email":"","orcid":"","institution":"Azienda Ospedaliera Universitaria Policlinico \"G. Martino\"","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Bonsignore","suffix":""},{"id":441192152,"identity":"b30196e4-c83e-433d-930a-86696d54e124","order_by":10,"name":"Maurizio Elia","email":"","orcid":"","institution":"Oasi Research Institute‐IRCCS","correspondingAuthor":false,"prefix":"","firstName":"Maurizio","middleName":"","lastName":"Elia","suffix":""},{"id":441192153,"identity":"470849f8-5a39-4253-b805-642fdd61f66b","order_by":11,"name":"Manuela Lo Bianco","email":"","orcid":"","institution":"University of Catania","correspondingAuthor":false,"prefix":"","firstName":"Manuela","middleName":"Lo","lastName":"Bianco","suffix":""},{"id":441192154,"identity":"e733c4be-59e5-424b-8a90-1a13c03871ec","order_by":12,"name":"Agata Polizzi","email":"","orcid":"","institution":"University of Catania","correspondingAuthor":false,"prefix":"","firstName":"Agata","middleName":"","lastName":"Polizzi","suffix":""},{"id":441192155,"identity":"8b3ff2b7-798a-47d4-822e-5c43b0fa5bbc","order_by":13,"name":"Martino Ruggieri","email":"","orcid":"","institution":"University of Catania","correspondingAuthor":false,"prefix":"","firstName":"Martino","middleName":"","lastName":"Ruggieri","suffix":""}],"badges":[],"createdAt":"2025-01-10 13:23:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5804009/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5804009/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-04718-6","type":"published","date":"2025-06-27T15:57:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80580928,"identity":"5622be08-fd50-423c-8ed1-75aa9ca852ff","added_by":"auto","created_at":"2025-04-14 23:18:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":146936,"visible":true,"origin":"","legend":"\u003cp\u003eGenealogic tree of the three large families reported in the present study. A) A family affected by mutation c.1498T\u0026gt;C of \u003cem\u003eTSC1\u003c/em\u003e; B) A family showing c.1096 G\u0026gt;T mutation of \u003cem\u003eTSC2\u003c/em\u003e; C) A large family affected by mutation c.3693_3696delGTCT of \u003cem\u003eTSC2\u003c/em\u003e. Patients affected by TSC are marked in black.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5804009/v1/a817e1ab2522591416d12ecc.png"},{"id":85686062,"identity":"c510d368-6f1e-40ff-bf2b-b527b5242327","added_by":"auto","created_at":"2025-06-30 16:02:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1155659,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5804009/v1/3870e444-6c80-43ec-a38f-52e564a746f8.pdf"},{"id":80579743,"identity":"6582d327-c071-4c12-a13b-5c7328053dbf","added_by":"auto","created_at":"2025-04-14 23:10:25","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":30324,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5804009/v1/777906f2fa3fe5b3b7daceca.xlsx"},{"id":80579745,"identity":"beb3c540-be0c-45a5-83a6-32f07877fcd2","added_by":"auto","created_at":"2025-04-14 23:10:25","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16990,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5804009/v1/c4bac2c54aaf69470233cf33.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetic Screening of Tuberous Sclerosis Complex in Sicily with a Focus on Neurological Manifestations","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTuberous Sclerosis Complex (TSC) (OMIM #191100; #613254) is a rare genetic disorder affecting approximately 1:6000-1:10,000 live births [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. First described by de Bourneville, TSC manifests with a wide clinical spectrum, including cutaneous, cardiac, renal, and neurological involvement, alongside neuropsychiatric manifestations known as TAND (Tuberous Sclerosis-Associated Neuropsychiatric Disorders) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The disease is caused by pathogenic heterozygous mutations in either the \u003cem\u003eTSC1\u003c/em\u003e gene (OMIM *605284) on chromosome 9 or the \u003cem\u003eTSC2\u003c/em\u003e gene (OMIM *191092) on chromosome 16 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. About two-thirds of cases result from \u003cem\u003ede novo\u003c/em\u003e pathogenic variants, especially for the \u003cem\u003eTSC2\u003c/em\u003e gene, while familial cases demonstrate equal distribution of variants between the two genes. Notably, 7.4% of patients meeting clinical diagnostic criteria lack identifiable mutations in \u003cem\u003eTSC1\u003c/em\u003e or \u003cem\u003eTSC2\u003c/em\u003e through conventional genetic testing, with somatic mosaicism and intronic mutations implicated in these cases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The hamartin-tuberin complex, encoded by \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e, acts as a negative regulator of the mechanistic target of rapamycin (mTOR) pathway. Dysfunction in this complex due to inactivating mutations leads to constitutive mTOR activation, resulting in cellular hyperproliferation and aberrant growth [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Clinical diagnostic criteria for TSC, first established in 2012 and revised in 2021, now encompass 11 major and seven minor features [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Histopathologically, TSC is marked by benign tumors (hamartomas) in multiple organs and cortical brain lesions termed \"tubers\", which disrupt synaptic networks and contribute to the disorder's neurological manifestations. Despite nearly complete penetrance, the clinical presentation of TSC is highly variable. Neurological symptoms such as epilepsy, cognitive impairment, and autism spectrum disorder (ASD) are predominant and significantly impact patients\u0026rsquo; quality of life [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Recent studies, including data from the TOSCA registry, consistently show that \u003cem\u003eTSC2\u003c/em\u003e mutations are associated with more severe clinical phenotype than \u003cem\u003eTSC1\u003c/em\u003e mutations [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This study analyzes the genotype-phenotype relationships in a Sicilian cohort, and evaluates the alignment of these findings with global research, with particular regard to epilepsy and neurological comorbidities.\u003c/p\u003e"},{"header":"PATIENTS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and Clinical Assessment\u003c/h2\u003e \u003cp\u003eThis study included 81 patients (mean age 26 years, range 2\u0026ndash;71) from four Italian centers (Unit of Pediatric Clinic and Unit of Rare Diseases of the Nervous System in Pediatric Age \u0026ndash; \"Policlinico-San Marco\u0026rdquo; University Hospital, Catania; Clinical and Instrumental Neurology and Neurophysiopathology Unit \u0026ndash; I.R.C.C.S. Oasi Troina; Regional Reference Center for Rare Genetic and Chromosomal Diseases \u0026ndash; Maternal and Child Department, \"Villa Sofia-Cervello\" Hospital, Palermo; Child Neuropsychiatry Unit, Santa Marta and Santa Venera Hospital, Acireale). Patients must present with a definite clinical diagnosis of TSC and a confirmed genetic alteration in \u003cem\u003eTSC1\u003c/em\u003e or \u003cem\u003eTSC2\u003c/em\u003e, including pathogenic and likely pathogenic variants, encompassing exonic mutations. Patients harboring intronic mutations with unknown or unpredictable effects at the protein level were included only if they had a definitive diagnosis of TSC, established according to the revised 2021 diagnostic criteria. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Retrospective analysis used data from existing medical records, supplemented by additional studies and follow-up during the period of the study. Collected data covered clinical, genetic, and radiological information. Regarding the epilepsy phenotype, for each patient data included age at onset (in months), seizure type, frequency (seizures per week, averaged over the most recent 12-month period), EEG findings, and treatment history. Seizure outcome was categorized based on response to treatment as follows: remission (\u0026gt;\u0026thinsp;75% reduction in seizure frequency), good control (50\u0026ndash;75%), partial control (25\u0026ndash;50%), and drug-resistant epilepsy (\u0026lt;\u0026thinsp;25%). Data were obtained through clinical records and follow-up evaluations. Classifications were made in accordance with standard clinical definitions. All patients had performed in the last three years an EEG, a brain MRI and a neuropsychiatric evaluation. Clinical investigation included a review of medical history and a careful physical examination by clinicians experienced in the manifestations of TSC. Brain MRI reports were collected and analyzed to identify specific patterns associated with cortical tubers, subependymal nodules (SENs), sub-ependymal giant cell astrocytoma (SEGA), and radial bands. Details on clinical signs and symptoms were obtained from the physicians who were sent a standardized clinical evaluation form. Members of the family were consulted when necessary. In addition, a range of standardized and validated psychological measures were used to assess intellectual level, autistic disorder, anxiety, depression, and behavior disorders. All manifestations were graded according to a binary scale (present or absent), but several features were graded in severity using numbers and words (characteristics of seizure, drug resistance, severity of intellectual disability, for example). Although complete phenotype information was collected, neurological and neuropsychiatric characteristics were the primary focus of analysis (supplementary tables 1 and 2). All TSC patients included in the study provided informed consent and had molecular diagnostic analysis for variants in \u003cem\u003eTSC1\u003c/em\u003e or \u003cem\u003eTSC2\u003c/em\u003e. The study protocol was approved by the Ethical Committee of the University of Catania, Italy. All methods were performed in accordance with the relevant guidelines and regulations\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMutational Analysis\u003c/h3\u003e\n\u003cp\u003eGenetic analysis was performed to identify pathogenic variants in \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e using a combination of multiplex ligation-dependent probe amplification (MLPA), denaturing high-performance liquid chromatography (DHPLC), and next-generation sequencing (NGS).\u003c/p\u003e \u003cp\u003eMLPA (MRC-Holland, SALSA P124/P046) was used to detect large deletions or duplications in \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e, while DHPLC was initially employed as a screening method to identify potential sequence variants, which were subsequently confirmed by Sanger sequencing.\u003c/p\u003e \u003cp\u003eNext-generation sequencing (NGS) was the preferred approach for patients born after 2016, allowing for a more comprehensive mutation screening. Targeted NGS was performed using a custom gene panel covering the entire coding regions and exon-intron boundaries of \u003cem\u003eTSC1\u003c/em\u003e (9q34.13) and \u003cem\u003eTSC2\u003c/em\u003e (16p13.3). The target region encompassed all exons and at least 20 bp of flanking intronic sequences to detect splicing-affecting variants. Sequencing was conducted on the Illumina MiSeq/NextSeq platform, with a mean coverage of \u0026gt;\u0026thinsp;200\u0026times;, ensuring a minimum of 30\u0026times; for reliable variant calling.\u003c/p\u003e \u003cp\u003eRaw sequence reads were processed using BWA-MEM for alignment to the hg19/hg38 reference genome, and variant calling was performed using GATK HaplotypeCaller. Identified variants were annotated using ANNOVAR, with pathogenicity assessed based on ClinVar, gnomAD, and ACMG guidelines. Large genomic deletions or duplications not detected by NGS were confirmed by MLPA.\u003c/p\u003e \u003cp\u003ePatients without identified pathogenic variants in \u003cem\u003eTSC1\u003c/em\u003e or \u003cem\u003eTSC2\u003c/em\u003e and not meeting the diagnostic criteria for Tuberous Sclerosis Complex were excluded from genotype-phenotype correlations.\u003c/p\u003e\n\u003ch3\u003eNeuropsychological evaluation\u003c/h3\u003e\n\u003cp\u003eNeuropsychiatric characteristics and cognitive impairment were evaluated using standardized neuropsychological tests administered by trained clinicians. Cognitive functioning was assessed using the Wechsler Preschool and Primary Scale of Intelligence (WPPSI-III or WPPSI-IV) for children aged 2 years to 7 years, and Wechsler Intelligence Scale for Children (WISC-IV) or the Wechsler Adult Intelligence Scale (WAIS-IV), depending on patient age. Full-scale IQ (FSIQ) was classified according to standard cut-off values: Normal cognitive function: FSIQ\u0026thinsp;\u0026ge;\u0026thinsp;85; Borderline cognitive function: FSIQ 70\u0026ndash;84; Mild intellectual disability (ID): FSIQ 55\u0026ndash;69M; Moderate ID: FSIQ 40\u0026ndash;54; Severe ID: FSIQ\u0026thinsp;\u0026lt;\u0026thinsp;40. For neuropsychiatric comorbidities, the presence of autism spectrum disorder (ASD) was determined using the Autism Diagnostic Observation Schedule-2 (ADOS-2) and Autism Diagnostic Interview-Revised (ADI-R), with classification based on DSM-5 criteria. Attention-deficit/hyperactivity disorder (ADHD) was diagnosed using the Conners Rating Scale and DSM-5 clinical criteria. Behavioral disorders, including oppositional defiant disorder (ODD) and anxiety disorders, were assessed using the Child Behavior Checklist (CBCL) and clinical interviews. For sleep disorders, a structured sleep history was taken, and the Pittsburgh Sleep Quality Index (PSQI) and Sleep Disturbance Scale for Children (SDSC) were used to classify sleep disturbances.\u003c/p\u003e \u003cp\u003e Specific Learning Difficulties (SLD), including dyslexia, dyscalculia, and dysgraphia, were assessed using standardized academic achievement tests appropriate for age and national guidelines. The following assessments were employed: \u003cb\u003eReading and dyslexia\u003c/b\u003e: Batteria per la Dislessia e Disortografia Evolutiva (DDE-2) for Italian-speaking children. \u003cb\u003eMathematics and dyscalculia\u003c/b\u003e: Test AC-MT (Test di Abilit\u0026agrave; di Calcolo e Matematica) for evaluating mathematical abilities. \u003cb\u003eWriting and dysgraphia\u003c/b\u003e: BVSCO-2 (Batteria per la Valutazione della Scrittura e della Competenza Ortografica) for assessing handwriting and spelling difficulties.\u003c/p\u003e \u003cp\u003eAll assessments were conducted at specialized centers with expertise in TSC, ensuring standardized evaluation across the cohort.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eQuantitative data were analyzed using Student\u0026rsquo;s t-test, while categorical variables were assessed using the Chi-square test. Statistical significance was determined for variables including age, onset of epilepsy, and frequencies of neurological manifestations. Percentages and proportions were calculated for categorical data, with significance assessed through a 2x2 contingency table analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eTSC1\u003c/b\u003e \u003cb\u003ePatients\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eTSC1\u003c/em\u003e group included 31 patients (15 men, 16 women), with an average age of 25.1 years (range: 2\u0026ndash;60 years). Of these, 45.1% had inherited mutations, while 54.9% were sporadic. \u003cem\u003eTSC1\u003c/em\u003e mutations were widely distributed along all the gene, involving in particular exons 4, 5, 6, 10, 15, 17, 18, 21 and 22. One patient presented an intronic variant (intron 1). Mutations were intragenic deletions (48.4%), nonsense (38.7%), missense (9.6%), and in one case a duplication (3.2%) (supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among these patients, six came from the same family and were affected by the already reported c.1498C\u0026thinsp;\u0026gt;\u0026thinsp;T, p.(Arg500Ter) mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eTSC1\u003c/em\u003e patients were affected by different types of seizures, differing in EEG findings, clinical course, age of onset and type of seizures. Epileptic seizures were present in 64.5% of patients, with onset averaging at 35.4 months. Seizure types were both generalized and focal crisis and some patients experienced multiple seizure types throughout life. More in detail, two patients (6.4%) presented infantile spasms, 12 (38.7%) had focal crises (3 with impaired awareness), 6 (19.3%) patients experienced generalized tonic clonic seizures, 4 (12.9%) suffered absence, one (3.2%) presented gelastic seziures, one (3.2%) reported seizures with fever, three (9.6%) suffered hypertonic crisis and one (3.2%) experienced atonic crisis. Seizure frequency ranged from 1 per lifetime to 28 per week (mean: 10.4 weeks, SD: 9.2). Severity and duration of crisis varied among patients and in the same patient. EEG abnormalities included spikes, waves, and hypsarrhythmia. Particularly, 11 patients (35.5%) had normal EEG, 1 (3.2%) presented hypsarrhythmia, 13 (41.9%) were reported to have spikes and waves (unilateral or bilateral), 11 (35.5%) had synchronous or asynchronous focal spikes, 1 (3.2%) had \u0026ldquo;arceau-like\u0026rdquo; pattern, 4 (12.9%) presented theta waves pattern. Epilepsy course varied from spontaneous remission to drug resistance, with valproic acid and carbamazepine being the most common treatments: 10 out of 20 patients (50%) achieved remission (complete response), while one (5%) had good response, 2 (10%) partial response, and the other and other 7 (35%) showed a drug-resistant epilepsy.\u003c/p\u003e \u003cp\u003eCognitive impairment levels of \u003cem\u003eTSC1\u003c/em\u003e patients ranged from mild to severe: 51.6% had normal cognitive functions, 9.7% had cognitive function near lower limit (borderline), 19.3% presented mild intellectual disability, 3.2% suffered from moderate intellectual disability and 16.1% had severe cognitive deficit. Autism spectrum disorder (ASD) was observed in four patients (12.9%), with only one patient showing Level 2 autism. Behavioral disorders (e.g., ADHD, anxious depressive syndrome, borderline personality) affected 8 patients (25.8%), while 12 (38.7%) experienced learning disabilities. Sleep disorders were reported in only 9.7%. Brain abnormalities of \u003cem\u003eTSC1\u003c/em\u003e group included cortical tubers (67.8% with \u0026gt;\u0026thinsp;5 tubers, mean size 8 mm) and subependymal nodules (51.6% with \u0026gt;\u0026thinsp;5 SENs, variable in size). Subependymal giant cell astrocytomas (SEGAs) were rare, found in only 2 patients (6.5%) probably related to a more severe genotype (non-sense mutations). White matter abnormalies and cerebral cysts were uncommon in our group of \u003cem\u003eTSC1\u003c/em\u003e patients, only 1 patient (3.2%) carrying the nonsense mutation c.2293C\u0026thinsp;\u0026gt;\u0026thinsp;T, had many cerebral cysts.\u003c/p\u003e \u003cp\u003eSome mutations were associated with specific clinical patterns. The mutation c.1888_1891delAAAG (found in two patients) was associated in both patients with many bilateral tubers and SENs, while epilepsy and neuropsychiatric disorders were heterogeneous, suggesting a potential role of other modifying factors. Mutations c.1004delC and c.2111_2112delAT were linked to minimal neurological symptoms and a good quality of life, indicating a milder impact. The c.1498T mutation (associated to a common specific EEG pattern and many brain hamartomas) was found in 6 patients of the same family (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and five of them showed the same EEG pattern (sharp theta waves and spikes in the left temporal lobe or bilateral). Notably, in this family, not all the patient showed epilepsy, and some of those affected were partial responders to drugs, while other drug-resistant. Cognitive functions and behavioral disorders were variable, with different grade of intellectual disability (absent to moderate/severe)\u003c/p\u003e \u003cp\u003e \u003cb\u003eTSC2\u003c/b\u003e \u003cb\u003ePatients\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis study analyzed 50 patients with \u003cem\u003eTSC2\u003c/em\u003e gene mutations, comprising 21 males (42%) and 29 females (58%), with a mean age of 27.1 years (SD\u0026thinsp;=\u0026thinsp;18.6, range 2\u0026ndash;71 years). Mutations were classified as familial (23 cases, 46%) or \u003cem\u003ede novo\u003c/em\u003e (27 cases, 54%) and showed extensive distribution across exons and included deletion (60%), nonsense (26%), missense (10%), and splicing mutations (6%). Furthermore, 4 intronic deletions were detected in this group (one spanning from intron 1 to exon 8), as well as a somatic mosaicism c.1373C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Arg1200Trp), involving 11.4% of the patient\u0026rsquo;s genome.\u003c/p\u003e \u003cp\u003eAmong these patients, two large families were included: one (5 members) harbored the c.1096G\u0026thinsp;\u0026gt;\u0026thinsp;T; (p.Glu366*) mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The other one comprised 16 members (among the 50 known members of this family), and presented the already reported c.3693_3696delGTCT (p.Ser1232Thrfs*92) mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eOf the 50 patients, 36 (72%) experienced seizures, with a mean onset age of 29.45 months (SD\u0026thinsp;=\u0026thinsp;66.3, range one month to 23 years). Seizure types varied, with 32% experiencing infantile spasms, 30% focal seizures (16% with impaired awareness) and 22% generalized tonic-clonic seizures. The frequency and severity of seizures ranged widely, with patients experiencing between 2 lifetime episodes to as many as 42 episodes per week (mean: 18.3, SD: 24.5). EEG patterns were varied, including theta waves, spikes, hypsarrhythmia, and focal spikes. Eighteen patients (36%) had normal EEG results, while focal and generalized spikes were more frequently observed. EEG patterns fluctuated over time within individual patients. The clinical course of epilepsy in \u003cem\u003eTSC2\u003c/em\u003e group of patients was variable, going from remission to multidrug resistance. Different antiseizures medications were used, including valproic acid, vigabatrin, carbamazepine, ACTH, topiramate, phenobarbital, levetiracetam, oxcarbazepine, lamotrigine, everolimus. Among them the most used were valproic acid and carbamazepine. \u003cem\u003eTSC2\u003c/em\u003e patients had been divided according to drug response into complete responder, (13 out of 36 patients- 36.1%), good responders (4 patients, 11%), partial responders (6 patients, 16.6%) and non-responders (9 patients, 25%).\u003c/p\u003e \u003cp\u003eCognitive impairment was prevalent among \u003cem\u003eTSC2\u003c/em\u003e patients, with 24%, 10%, and 24% exhibiting mild, moderate, and severe intellectual disability, respectively, while 42% displaying normal cognitive function. ASD was diagnosed in 12% of patients, while 48% had learning disabilities. Behavioral issues were present in 30% of patients, predominantly ADHD (20%), irritability (10%), and oppositional behavior (10%). It is necessary to underline that many patients presented more than one behavior disorder. Sleep disorders were rare, with only a few cases of insomnia or enuresis reported. MRI analyses revealed that 10% of \u003cem\u003eTSC2\u003c/em\u003e patients had no cortical tubers, while 74% had multiple tubers (\u0026gt;\u0026thinsp;5). The average tuber sizes was 17 mm (SD\u0026thinsp;=\u0026thinsp;18.3mm), with some patients presenting cerebellar or cystic tubers. Subependymal nodules (SENs) were identified in 86% of patients, with dimensions varying widely (average size\u0026thinsp;=\u0026thinsp;9.4mm, SD\u0026thinsp;=\u0026thinsp;0.9mm). SEGAs were less common, found in 14% of patients. The average size of SEGAs was 1.3cm (SD\u0026thinsp;=\u0026thinsp;15mm), ranging from 1cm to 1.5cm. In our cohort SEGAs were associated with different genotypes, in particular the mutations c.3094C\u0026thinsp;\u0026gt;\u0026thinsp;T, c.5201_5216dupATATCTACCCCTCCAA and c.1238_1285delCCT. One patient harboring 2 SEGAs carried a large intragenic deletion (spanning from exon 3 to 9), that could explain the increased number of SEGAs and a more severe neuropsychiatric and epileptic phenotype. Radial bands were noted in 12% of cases, mostly in parietal and temporal regions. Most patients (74%) had no white matter abnormalities, the remaining cases ranged from multiple to unspecified abnormalities. Only 3 patients (6%) had brain cysts and 2 (4%) had atrophy or hypoplasia of the corpus callosum.\u003c/p\u003e \u003cp\u003eRegarding the specific mutations c.3693_3696delGTCT, (p.Ser1232Thrfs*92), it was found in one of the biggest family of TSC patients in Italy. The family is made up of at least 43 members (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), of whom at least 25 members are affected by tuberous sclerosis. Complete information about genotype and phenotype of all the members was impossible to collect for many reasons (for example, some of them died without certain diagnosis, others refused clinical and genetic investigations, data were stored in different hospitals): for this reason, a total of 16 family members have been included in this study. Although they all carry the same mutation, an intrafamilial variable clinical presentation was observed, going from mild cutaneous phenotype to severe mental retardation and drug-resistant epilepsy. Nine patients from this family presented epilepsy. The median age of onset of epilepsy was 6 years old, ranging from birth to 23 years. Types of seizures and frequency were variable: infantile spasms, tonic clonic, myoclonic, focal, febrile crisis are only few examples. The most frequent were infantile spasms, tonic clonic and focal crises with impaired awareness. Regarding the EEG, the most frequent patterns were hypsarrhythmia and focal spikes (with secondary generalization) in the left frontal temporal lobes: it must be underlined that all the normal EEGs found in \u003cem\u003eTSC2\u003c/em\u003e epileptic patients were observed in this family. Drug response was different among the members of this family, and the biggest group is made up of good/partial responders, although there were 4 cases of remission. All the levels of severity of cognitive impairment were present in this family, although the most frequent was mild intellectual disability. ASD was rare in this family and none of them reached the diagnostic criteria for confirmed diagnosis, only some of them had a sub-clinical form of ASD with limited social difficulties. Learning disability was present mainly in patients with moderate-severe intellectual disability, who were the minority of the family. The most frequent behavior disorders were ADHD and oppositional disorders. Sleep disorders were basically absent. Many bilateral tubers and SENs were reported in all the members of the family, while none of them presented SEGAs. Cerebral cysts and white matter abnormalities were absent, while some of them presented radial bands. To sum up the common characteristics of this family members were multiple tubers and SENs, absence of SEGAs, attention deficit disorder, mild mental retardation (that could explain why they managed to reproduce), partial epilepsy control, infantile spasms and partial complex crisis.\u003c/p\u003e \u003cp\u003eThe mutation c.1096G\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Glu366Ter) was present in another family (patients 69,70,71,72,73) (respectively a father and his two sons and two daughters). The types of seizures described in this family were focal with impaired awareness and infantile spasms. All patients also shared severe intellectual and learning disability. ASD, behavior and sleep disorders were absent. Brain MRI reports revealed the presence of few to many tubers and SEN, but no-one showed SEGA.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTSC1\u003c/b\u003e \u003cb\u003evs.\u003c/b\u003e \u003cb\u003eTSC2\u003c/b\u003e \u003cb\u003ePatients\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe study cohort included 81 patients, divided into \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e groups. Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e summarize the comparison between the two groups and the statistical data.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMain Clinical Characteristics of the two groups of patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTSC1\u003c/em\u003e patients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTSC2\u003c/em\u003e patients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of Patients\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31(38,3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50(61.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemales\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16(51.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29(58%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage Age (Standard Dev) [Years]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.1(16.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.1(18.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge Range [Years]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u0026ndash;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u0026ndash;71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDe Novo Mutations\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18(58.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27(54%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFamilial Mutations\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13(41.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23 (46%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntragenic Deletions\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15(48.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30(60%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntragenic Duplications\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1(3.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2(3.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMissense Mutations\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3(9.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5(10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNonsense Mutations\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12(38.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13(26%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEpilepsy and Neuropsychiatric comorbidities of the two groups of patients (included if at least 5 individuals presented the specific feature) * Patient 9, 44 and 49 - experiencing only one to two seizures in their lifetime \u0026ndash; are excluded from this range.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTSC1\u003c/em\u003e patients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTSC2\u003c/em\u003e patients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSeizures\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean Age at epilepsy onset (SD) [Months]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.4(25.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.45(66.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge range of epilepsy onset [Months]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1-276\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatients Without Seizures\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11(35.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14(28%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInfantile Spasms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2(6.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16(32%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFocal seizures\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12(38.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15(30%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFocal seizures with impaired awareness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3(9.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8(16%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGeneralized tonic-clonic seizures\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6(19.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11(22%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsence Seizures\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4(12.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4(8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypertonic seizures\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3(9.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5(10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeizure Frequency (SD) [episodes per week]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.4 (9.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.3 (24.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS (0.089)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRange of seizures per week\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;28*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026ndash;42*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRemission of Seizures [\u0026gt;\u0026thinsp;75% of reduction]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10(50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13(36.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePartial/ good response (25 to 74.9% of reduction)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3(15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10(27.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrug Resistant patients\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7(35%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9(25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEEG findings\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11(35.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 (36%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypsarrhythmia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1(3.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5(10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBilateral Spikes and Waves Pattern\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7(22.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12(24%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFocal Anomalies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8(25.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15(30%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTheta Waves Pattern\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4(12.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6(12%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNeuropsychiatric comorbidities\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal Cognitive Functions\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16(51.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21(42%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMild Intellectual disability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6(19.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12(24%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModerate Intellectual disability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1(3.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5(10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSevere Intellectual disability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5(16.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12(24%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatients Without Autism Spectrum Disorder\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27(87.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44(88%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAutism Level 1 and 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4(12.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6(12%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo learning Disability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19(61.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26(52%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLearning Disability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12(38.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24(48%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsence of Behavior Disorders\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23(74.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35(70%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eADHD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3(9.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10(20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIrritability And Agitation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3(9.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5(10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOppositional Disorder/Aggression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5(10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsence of Sleep disorders\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28(90%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48(96%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMRI finding of the two groups of patients. SD: Standard Deviation; SEGA: Subependymal giant cell astrocytoma.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTSC1\u003c/em\u003e patients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTSC2\u003c/em\u003e patients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTubers\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsence of tubers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (16%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 to 5 tubers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (16.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (14%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMore than 5 tubers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21 (67.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36 (72%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean Size Of Tubers (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 mm (3.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17mm (18.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubers Dimensions range (95% C.I.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026ndash;20 mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u0026ndash;30 mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSubependymal nodules\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsence of Subependymal nodules\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5(16.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11(22%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 to 5 Subependymal nodules\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10(32.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2(4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMore than 5 subependymal nodules\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16(51.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31(62%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage size of subependymal nodules (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.87 mm (2.09)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.4 mm (0.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubependymal nodules dimensions range (95% C.I.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-10mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026ndash;12 mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSubependymal giant-cell astrocytomas\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatients without SEGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29 (93.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43 (86%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 SEGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (6.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (12%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 or more SEGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatients underwent SEGA surgery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (57%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage size Of SEGAs (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.1 cm (0.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.3 cm (0.15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSEGA dimensions range (95% C.I.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 -1.2cm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1-1.5 cm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRadial Bands\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePresence of Radial Bands\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 (38.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (12%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 Radial Band\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (9.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 or more Radial Bands\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 (29%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCerebral Cysts\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsence of Cerebral Cysts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30 (96.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47 (94%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 Cerebral Cyst\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 or more Cerebral Cysts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (3.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eWhite Matter Abnormalities\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsence of White Matter Abnormalities\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22 (71%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37 (74%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLimited White Matter Abnormalities\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (12.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiffuse White Matter Abnormalities\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (16.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (16%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe proportion of \u003cem\u003eTSC2\u003c/em\u003e patients (61.7%) was higher than \u003cem\u003eTSC1\u003c/em\u003e ones (38.3%). There was not statistically significant difference between \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e groups with respect to sex (\u003cem\u003eTSC1\u003c/em\u003e: male 48.4%-female 51.6%; \u003cem\u003eTSC2\u003c/em\u003e: male 42%-female 58%) and to the mean age of patients. In our cohort de novo and familial mutations were proportionally distributed between the two groups. There was no statistically significant difference for types of mutations, which included intragenic deletions, duplications, missense and nonsense mutations. \u003cem\u003eTSC2\u003c/em\u003e patients exhibited a high variability in clinical manifestations, seizure types, cognitive outcomes, and behavioral disorders. \u003cem\u003eTSC2\u003c/em\u003e group had a higher prevalence of epilepsy than \u003cem\u003eTSC1\u003c/em\u003e patients (72% vs. 64.5%, not statistically significant) and in particular of infantile spasms (p\u0026thinsp;=\u0026thinsp;0.016). About other type of seizures there was not any significant difference between the two groups: focal crisis (p\u0026thinsp;=\u0026thinsp;0.3651), tonic clonic crisis (p\u0026thinsp;=\u0026thinsp;0.94), gelastic crisis (p\u0026thinsp;=\u0026thinsp;0.72), focal dyscognitive crisis (p\u0026thinsp;=\u0026thinsp;0.41), febrile crises (p\u0026thinsp;=\u0026thinsp;0.38), atonic crisis (p\u0026thinsp;=\u0026thinsp;0.87), hypertonic crisis (p\u0026thinsp;=\u0026thinsp;0.96), epileptic status (p\u0026thinsp;=\u0026thinsp;0.25) and myoclonic crisis (p\u0026thinsp;=\u0026thinsp;0.16). There was no statistically significant difference in the number of weekly episodes between the two groups, but a tendency of \u003cem\u003eTSC2\u003c/em\u003e patients to present more seizure per week (p\u0026thinsp;=\u0026thinsp;0.089). Regarding EEG, a normal pattern was found in the same proportion of \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e patients. The only appreciable difference between \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e patients was in the prevalence of hypsarrhythmia (higher in \u003cem\u003eTSC2\u003c/em\u003e patients, even if not statistically significant). Both groups showed similar responses to antiseizure medications, with no significant difference in rates of remission, partial or good response, or drug-resistance. Cognitive and behavioral disorders were similarly distributed, although \u003cem\u003eTSC1\u003c/em\u003e patients had higher rates of borderline cognitive function, while \u003cem\u003eTSC2\u003c/em\u003e patients had more severe neuropsychiatric profiles compared to \u003cem\u003eTSC1\u003c/em\u003e (not statistically significant). Importantly, patients presenting remission from seizures were likely to have normal cognitive profile: among the \u003cem\u003eTSC1\u003c/em\u003e responders, 70% did not show any intellectual disability (90% considering also those mildly affected). Among \u003cem\u003eTSC2\u003c/em\u003e patients, 28.6% showing remission did not show any intellectual disability, and the ration increased to 78.6% if considering normal to mild intellectual disability.\u003c/p\u003e \u003cp\u003eTubers, SENs, and radial bands occurred at comparable frequencies, with \u003cem\u003eTSC2\u003c/em\u003e patients slightly more likely to have multiple and larger tubers (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and larger SEN (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while \u003cem\u003eTSC1\u003c/em\u003e patients to have radial (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and multiple bands (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study aimed to conduct a mutational screening of a cohort of Sicilian TSC patients, compare the clinical features of \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e groups, and described four new potential genotype-phenotype correlations.\u003c/p\u003e \u003cp\u003eThe cohort includes 81 patients, representing approximately 15% of the estimated TSC population in Sicily, assuming a prevalence of 1 in 8,000 individuals. This proportion suggests that our study provides a significant and representative sample, offering valuable insights into the genetic and phenotypic spectrum of the disease in this region. Given the focus on Sicily, it is relevant to underline that, despite the application of advanced molecular techniques, certain challenges may limit the full identification of TSC cases, including potential underdiagnosis due to disparities in healthcare access and variability in disease expression: patients with milder phenotypes can remain undiagnosed, particularly in cases where dermatological or renal manifestations are subtle, and epilepsy is absent or well-controlled. However, genetic testing and specialized care are becoming increasingly accessible across all Sicilian provinces, enhancing the identification and diagnosis of TSC cases. These factors highlight the need for continued efforts in optimizing TSC surveillance and diagnostic strategies in this population.\u003c/p\u003e \u003cp\u003eThe proportion of \u003cem\u003eTSC2\u003c/em\u003e patients was higher than \u003cem\u003eTSC1\u003c/em\u003e, consistent with literature indicating that approximately two-thirds of pathogenic variants are \u003cem\u003eTSC2\u003c/em\u003e, and one-third are \u003cem\u003eTSC1\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Gender distribution and mean age were similar between the two groups, in agreement with previous studies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eDe novo\u003c/em\u003e mutations represented 54.3% of the total, while familial mutations accounted for 45.6%. This differs from literature suggesting a greater disparity (approximately 2:1 \u003cem\u003ede novo\u003c/em\u003e vs familial cases), likely due to challenges in accurately reconstructing family histories in our cohort [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. There was no significant difference in the proportions of \u003cem\u003ede novo\u003c/em\u003e and familial mutations between the \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e groups (p\u0026thinsp;=\u0026thinsp;0.9413), differently from literature data that suggest a higher proportion of \u003cem\u003eTSC2\u003c/em\u003e mutations in sporadic cases [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The type of mutations \u0026ndash; deletions, duplications, missense, and nonsense \u0026ndash; were equally distributed, diverging from studies that describe distinct distributions for \u003cem\u003eTSC1\u003c/em\u003e (predominantly nonsense and deletions) and \u003cem\u003eTSC2\u003c/em\u003e (mainly frameshift, nonsense, and deletions) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This discrepancy may reflect our smaller sample size. However, some reports align with our findings, describing similar proportions of missense and other type of mutations in \u003cem\u003eTSC2\u003c/em\u003e patients [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsistent with previous studies, \u003cem\u003eTSC1\u003c/em\u003e patients had a lower probability of experiencing seizures compared to \u003cem\u003eTSC2\u003c/em\u003e patients, even without statistical significance (35.5% of \u003cem\u003eTSC1\u003c/em\u003e patients did not present seizures, vs. 28% of \u003cem\u003eTSC2\u003c/em\u003e patients) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, these last showed a significantly higher prevalence of infantile spasms (p\u0026thinsp;=\u0026thinsp;0.016), a finding aligned with earlier studies (19% in \u003cem\u003eTSC1\u003c/em\u003e vs. 41% in \u003cem\u003eTSC2\u003c/em\u003e) [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Interestingly, the age of epilepsy onset was similar (but patients with \u003cem\u003eTSC2\u003c/em\u003e mutations tended to exhibit a higher number of seizures per week), partially in line with literature that suggests an earlier onset and more frequent drug-resistant epilepsy in \u003cem\u003eTSC2\u003c/em\u003e [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This could be due to limitations in seizure quantification or the dynamic nature of drug responsiveness. About drug responsiveness, percentages of seizure remission, good or partial responders and drug-resistant patients were comparable in \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e group, even if \u003cem\u003eTSC1\u003c/em\u003e patients tend to show a higher ratio of seizures remission (50% vs. 36.1%), but a lower amount of partial or good responders (i.e. seizure reduction from 25 to 74.9%), which was higher in \u003cem\u003eTSC2\u003c/em\u003e patients (27.6% vs 15%). These results are somewhat in contrast with other studies, that clearly affirm that \u003cem\u003eTSC2\u003c/em\u003e patients suffer more severe and drug-resistant epilepsy [\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the same way, EEG findings were substantially similar in the two groups, with a higher proportion of \u003cem\u003eTSC2\u003c/em\u003e patients showing hypsarrhythmia, even if not statistically different.\u003c/p\u003e \u003cp\u003eWe found no significant difference in the severity of intellectual disability between \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e patients, although mild intellectual disability was more prevalent in \u003cem\u003eTSC1\u003c/em\u003e patients (p\u0026thinsp;=\u0026thinsp;0.225). This partially contrasts with reports associating more frequently \u003cem\u003eTSC2\u003c/em\u003e individuals with severe intellectual impairment, likely influenced by epilepsy severity and tuber burden (20% \u003cem\u003eTSC1\u0026ndash;51\u003c/em\u003e% \u003cem\u003eTSC2\u003c/em\u003e) [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Interestingly, specific \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e mutations have been linked to both mild and severe phenotypes, illustrating the heterogeneity within and between genotypes. Nevertheless, the association between \u003cem\u003eTSC2\u003c/em\u003e mutations and severe intellectual disability is not constantly present. Some \u003cem\u003eTSC2\u003c/em\u003e genotypes have been found to be associated with mild phenotype without cognitive impairment, such as c.1514G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.Arg505Gln), c.3598C\u0026thinsp;\u0026gt;\u0026thinsp;T (p. Arg1200Trp) and c.2714G\u0026thinsp;\u0026gt;\u0026thinsp;A (Arg905Gln). On the other hand, some specific \u003cem\u003eTSC1\u003c/em\u003e mutations have been associated with intellectual disability such as c.682C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Arg228Ter), c.1117C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Gln373Ter), c.2713C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Arg905Trp) and c.2713C\u0026thinsp;\u0026gt;\u0026thinsp;G (p.Arg905Gly) [\u003cspan additionalcitationids=\"CR34 CR35 CR36 CR37 CR38 CR39\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Furthermore, a different severity of intelligence deficit has been described in the same mutational group: for example, patients with a \u003cem\u003eTSC1\u003c/em\u003e variant in the tuberin interacting domain showed significantly lower IQ compared to other \u003cem\u003eTSC1\u003c/em\u003e variants. Similarly, mutations causing truncation of the proximal part of \u003cem\u003eTSC2\u003c/em\u003e or involving the hamartin-interacting domain cause more severe intellectual disability than small in-frame deletions in the distal part of the gene or missense mutations. These findings provide further evidence that different types and locations of \u003cem\u003eTSC1\u003c/em\u003e or \u003cem\u003eTSC2\u003c/em\u003e pathogenic variants may be associated with distinct neurocognitive phenotypes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImportantly, patients who experienced seizure remission were more likely to have a normal cognitive profile. Among \u003cem\u003eTSC1\u003c/em\u003e responders, 70% had no intellectual disability, and this proportion rose to 90% when including those with mild intellectual impairment. In the \u003cem\u003eTSC2\u003c/em\u003e group, 28.6% of patients with seizure remission had no intellectual disability, increasing to 78.6% when considering both normal cognitive profiles and mild intellectual disabilities. On the other hand, among the drug-resistant patients, intellectual disability was severe in 4 out 7 \u003cem\u003eTSC1\u003c/em\u003e drug-resistant patients (57.1%) and in 6 out of 9 (66.6%) \u003cem\u003eTSC2\u003c/em\u003e drug-unresponsive patients (see supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results are consistent with the literature and support the current view that preventing the development of drug resistance may help preserve or improve cognitive outcomes in patients with TSC [\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNo significant differences emerged in the prevalence of learning disabilities, ASD, or behavioral disorders, including ADHD, irritability, borderline personality, and sleep disturbances. These findings contrast with reports of higher ASD prevalence and behavioral challenges in \u003cem\u003eTSC2\u003c/em\u003e patients [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The lack of significant differences may reflect the small cohort size of patients presenting these neuropsychiatric conditions.\u003c/p\u003e \u003cp\u003eTuber prevalence was similar between groups, though \u003cem\u003eTSC1\u003c/em\u003e patients were more likely to have smaller tubers (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). This aligns with some studies but contrasts with reports of a higher cortical tuber burden in \u003cem\u003eTSC2\u003c/em\u003e (91% vs 76%) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. SENs, SEGAs, and white matter abnormalities were equally frequent, though \u003cem\u003eTSC1\u003c/em\u003e patients exhibited a higher presence of radial bands (p\u0026thinsp;=\u0026thinsp;0.011) and a more likely occurrence of more than two bands (p\u0026thinsp;=\u0026thinsp;0.028): this represents a previously unreported finding in patients with TSC.\u003c/p\u003e \u003cp\u003eThe main reviews published so far report that \u003cem\u003eTSC2\u003c/em\u003e patients usually present a more severe phenotype, for nearly all the features. However, there can be overlap in disease severity between the two groups, indeed many \u003cem\u003eTSC1\u003c/em\u003e patients were found to have severe neurocutaneous phenotype and some patients with \u003cem\u003eTSC2\u003c/em\u003e mutations had mild phenotype, representing exceptions to the general rule [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Therefore, in our opinion, clinicians should not strictly rely on genotype for clinical counseling and prognosis, unless a specific genotype-phenotype correlation is available.\u003c/p\u003e \u003cp\u003eIn this regard, genotype-phenotype correlations may be challenging in clinical practice, due to the high inter- and intra-familial phenotype variability, the age dependence onset of certain manifestations, the role of genetic and environmental modifiers, including cases of mosaicism, and the rare occurrence of specific (unknown) variants. Despite that, genotype\u0026ndash;phenotype correlation is essential for clinical management, prognosis and personalized treatments, and are currently used by the clinicians to oversee the outcome of TSC patients, especially in some specific cases.\u003c/p\u003e \u003cp\u003eBased on the genetic and clinical data from this study, we explored four tentative genotype\u0026ndash;phenotype associations in families with multiple affected individuals. However, given the marked intrafamilial phenotypic variability observed, these associations should not be interpreted as strict correlations, but rather as hypothesis-generating observations requiring further validation.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eTSC2\u003c/em\u003e mutation c.3693_3696delGTCT (p.Ser1232Thrfs*92) was found in a large family (43 members, 25 affected, 16 reported in the present study). The clinical presentation was variable, going from mild cutaneous phenotype to severe intellectual disability and drug-resistant epilepsy. The shared features included multiple tubers and SENs, ADHD, mild intellectual disability (which may explain the high reproductive rates in certain individuals), partial/good seizure control, infantile spasms and focal seizures with impaired awareness. Unfortunately, complete information about genotype and phenotype of all the members was impossible to collect for many reasons (some of them had died without certain diagnosis, others refused clinical and genetic investigations, some data were stored in different hospitals and clinics).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eTSC2\u003c/em\u003e mutation c.1096G\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Glu366Ter) was observed in five-member family, associated with severe intellectual disability and specific seizure types (focal crises with impaired awareness and infantile spasms).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eTSC1\u003c/em\u003e mutation c.1498T (p.Arg500Ter) identified in six members of a large family, could be associated to a distinct EEG pattern (sharp theta waves and spikes in the left temporal lobe or bilateral) and multiple brain tubers and SENs, but no SEGAs. Despite that, cognitive functions were variable and not every patient presented seizures, but those affected were partial responders.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eTSC1\u003c/em\u003e mutation c.2111_2112delAT (p.Tyr704Ter) was found in three patients of a small family, with a mild neurological phenotype, multiple bilateral tubers and SENs but no seizures or neuropsychiatric disorders.\u003c/p\u003e \u003cp\u003eIn addition to the reported observations that (sometime) phenotypes associated with \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e pathogenic variants overlap substantially, we did not always observe significant differences in relation to the type of gene mutation in our retrospective analysis. However, overall results and genotype-phenotype correlations observed in this study reinforce evidence from larger cohorts, particularly those indicating that \u003cem\u003eTSC2\u003c/em\u003e mutations result in a more severe clinical phenotype, than \u003cem\u003eTSC1\u003c/em\u003e mutations.\u003c/p\u003e \u003cp\u003eOur data reveal that \u003cem\u003eTSC2\u003c/em\u003e patients face a significantly higher burden of epilepsy and EEG abnormalities (in particular infantile spasms and hypsarrhythmia), supporting existing evidence that \u003cem\u003eTSC2\u003c/em\u003e mutations may drive hyperactivity within the mTOR pathway more forcefully than \u003cem\u003eTSC1\u003c/em\u003e, thereby increasing neural excitability [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Such findings underscore the importance of identifying TSC genotypes early, as \u003cem\u003eTSC2\u003c/em\u003e patients may benefit from proactive epilepsy management with vigabatrin, cannabidiol or mTOR-inhibitors [\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The higher rate of EEG abnormalities in \u003cem\u003eTSC2\u003c/em\u003e patients, including hypsarrhythmia and focal spikes, correlates with findings by Chu-Shore et al., who reported similar EEG profiles in \u003cem\u003eTSC2\u003c/em\u003e patients with refractory epilepsy [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. These EEG patterns, frequently associated with epileptic encephalopathies, suggest that tubers and associated neural dysplasia in \u003cem\u003eTSC2\u003c/em\u003e patients might extend beyond visible MRI lesions, potentially involving perituberal regions [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe neuropsychiatric profile of \u003cem\u003eTSC2\u003c/em\u003e patients, including a higher prevalence of ADHD and severe cognitive impairment, suggests that \u003cem\u003eTSC2\u003c/em\u003e mutations exert a greater impact on cognitive and behavioral domains, possibly through disruption of cortical networks involved in attention and executive function [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] Our data confirm findings from Bolton et al. and others who observed a correlation between \u003cem\u003eTSC2\u003c/em\u003e mutations and ASD severity, underscoring the need for early intervention strategies, particularly for \u003cem\u003eTSC2\u003c/em\u003e patients at risk of developmental delays and behavioral disturbances [\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese findings highlight the value of genotype-specific approaches in managing TSC patients. Given the severe phenotype associated with \u003cem\u003eTSC2\u003c/em\u003e mutations, early mTOR inhibitor therapy may be beneficial, not only for seizure control but also potentially for reducing neuropsychiatric symptoms, as suggested by recent clinical trials [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Furthermore, given the correlation between \u003cem\u003eTSC1\u003c/em\u003e and radial band prevalence, also \u003cem\u003eTSC1\u003c/em\u003e patients might benefit from neurodevelopmental monitoring, even in the absence of severe epilepsy, to address potential subclinical impacts on cognitive function.\u003c/p\u003e \u003cp\u003eWhile our findings align with existing literature, the limited cohort size necessitates caution when generalizing results. Further multicenter studies with larger sample sizes could provide a deeper understanding of rare features and refine genotype-specific treatment guidelines. Additionally, longitudinal studies are essential to evaluate the long-term impact of early genotype-informed interventions on neurodevelopmental and psychiatric outcomes in TSC patients.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThis study represents the first comprehensive TSC mutational analysis and genotype-phenotype correlation conducted in Sicilian TSC patients. Our findings reinforce the established association of \u003cem\u003eTSC2\u003c/em\u003e mutations with more severe neurological manifestations, including infantile spasms and related EEG abnormalities. Conversely, patients harboring \u003cem\u003eTSC1\u003c/em\u003e mutations showed a higher prevalence of radial bands, and milder cognitive involvement, emphasizing the nuanced heterogeneity of TSC.\u003c/p\u003e \u003cp\u003eThe proposed genotype-phenotype correlations highlight the variability within TSC manifestations and underscore the need for personalized approaches to management. Our results support early genetic testing to guide treatment strategies, particularly for \u003cem\u003eTSC2\u003c/em\u003e patients who may benefit from mTOR-inhibitor therapies and proactive epilepsy management.\u003c/p\u003e \u003cp\u003e Larger multicenter and longitudinal studies are essential to confirm these correlations, refine genotype-specific therapeutic guidelines, and evaluate long-term outcomes of early interventions. By leveraging genetic insights, clinicians can advance precision medicine in TSC, improving quality of life and clinical outcomes for affected patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e: Approvement of the Ethical committee of the University has been obtained, as well as consent to participate from patients and/or parents or legal guardians of minor patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eWritten informed consent for publication of this study was obtained from all adult patients and the parents or legal guardians of minor participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e: The datasets generated and/or analyzed during the current study are available in the ClinVar repository (https://www.ncbi.nlm.nih.gov/clinvar/) and can be retrieved using the following submission IDs: 15233733; 15233796; 15233928; 15233938; 15233951; 15233966; 15233980; 15233928; 15234011; 15234019; 15234029; 15234041; 15234045; 15234142; 15234050; 15234063; 15233951; 15234070; 15234109 15234148; 15234153; 15234159; 15234185; 15234192; 15234203; 15237360; 15234242; 15234212; 15234220; 15234224; 15234375; 15234222; 15234237; 15234362; 15237397; 15234207; 15234240; 15237421, which will be linked to public variant accession numbers upon final processing. The data can be accessed upon reasonable request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e: The authors report no conflict of interest in the subject presented by the present research\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: No funding has been received for the preparation of this article\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution\u003c/strong\u003e: ADP wrote the first draft of the manuscript; CD, ED and SS performed the literature research; MP, FC, RS, MV, AZ and MB followed-up the patients; ME and MLB validated the results; AP \u0026amp; MR critically revised the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: The Authors wish to thank the \u0026ldquo;Associazione Sclerosi Tuberosa\u0026rdquo; (AST) for its support; The Authors also wish to thank the Health Operational Plan (FSC 2014-2020) \u0026ldquo;Trajectory 4-Biotechnology, Bioinformatics and Pharmaceutical Development\u0026rdquo; entitled \u0026ldquo;PHARMA-HUB\u0026rdquo;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOsborne, J. P. et al. Epidemiology of tuberous sclerosis. \u003cem\u003eAnn. N Y Acad. Sci.\u003c/em\u003e \u003cb\u003e615\u003c/b\u003e, 125\u0026ndash;127 (1991).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG\u0026oacute;mez, M. R. et al. History of the tuberous sclerosis complex. \u003cem\u003eBrain Dev.\u003c/em\u003e \u003cb\u003e17 Suppl\u003c/b\u003e, 55\u0026ndash;57 (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrigo, F. et al. First descriptions of tuberous sclerosis by D\u0026eacute;sir\u0026eacute;-Magloire Bourneville (1840\u0026ndash;1909). \u003cem\u003eNeuropathology\u003c/em\u003e \u003cb\u003e38\u003c/b\u003e, 577\u0026ndash;582 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuschner, F. et al. Molecular EPISTOP, a comprehensive multi-omic analysis of blood from Tuberous Sclerosis Complex infants age birth to two years. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (1), 7664 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKandt, R. S. et al. Linkage of an important gene locus for tuberous sclerosis to a chromosome 16 marker for polycystic kidney disease. \u003cem\u003eNat. Genet.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, 37\u0026ndash;41 (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTyburczy, M. E. et al. Mosaic and intronic mutations in TSC1/TSC2 explain the majority of TSC patients with no mutation identified by conventional testing. \u003cem\u003ePLoS Genet.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, e1005637 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe, Z. et al. Mosaicism in tuberous sclerosis complex: lowering the threshold for clinical reporting. \u003cem\u003eHum. Mutat.\u003c/em\u003e \u003cb\u003e43\u003c/b\u003e, 1956\u0026ndash;1969 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInoki, K. et al. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. \u003cem\u003eNat. Cell. Biol.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 648\u0026ndash;657 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNorthrup, H. et al. Updated international tuberous sclerosis complex diagnostic criteria and surveillance and management recommendations. \u003cem\u003ePediatr. Neurol.\u003c/em\u003e \u003cb\u003e123\u003c/b\u003e, 50\u0026ndash;66 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuratolo, P. et al. Neurological and neuropsychiatric aspects of tuberous sclerosis complex. \u003cem\u003eLancet Neurol.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 733\u0026ndash;745 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinto, A. L. R. et al. Neurological manifestations of tuberous sclerosis complex: the importance of early diagnosis. \u003cem\u003eArq. Neuropsiquiatr.\u003c/em\u003e \u003cb\u003e80\u003c/b\u003e, 983\u0026ndash;984 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNabbout, R. et al. Historical Patterns of Diagnosis, Treatments, and Outcome of Epilepsy Associated With Tuberous Sclerosis Complex: Results From TOSCA Registry. \u003cem\u003eFront. Neurol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 697467 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDabora, S. L. et al. Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. \u003cem\u003eAm. J. Hum. Genet.\u003c/em\u003e \u003cb\u003e68\u003c/b\u003e, 64\u0026ndash;80 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNorthrup, H., Koenig, M. K. \u0026amp; Pearson, D. A. Tuberous sclerosis complex. Tuberous Sclerosis Complex.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeron, A. et al. Genetics, genomics, and genotype-phenotype correlations of TSC: insights for clinical practice. \u003cem\u003eAm. J. Med. Genet. C Semin Med. Genet.\u003c/em\u003e \u003cb\u003e178\u003c/b\u003e, 281\u0026ndash;290 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuratolo, P. et al. Tuberous sclerosis. \u003cem\u003eHandb. Clin. Neurol.\u003c/em\u003e \u003cb\u003e111\u003c/b\u003e, 323\u0026ndash;331 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLangkau, N. et al. TSC1 and TSC2 mutations in tuberous sclerosis, the associated phenotypes and a model to explain observed TSC1/TSC2 frequency ratios. \u003cem\u003eEur. J. Pediatr.\u003c/em\u003e \u003cb\u003e161\u003c/b\u003e, 393\u0026ndash;402 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSancak, O. et al. Mutational analysis of the TSC1 and TSC2 genes in a diagnostic setting: genotype\u0026ndash;phenotype correlations and comparison of diagnostic DNA techniques in tuberous sclerosis complex. \u003cem\u003eEur. J. Hum. Genet.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 731\u0026ndash;741 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAu, K. S. et al. Genotype/phenotype correlation in 325 individuals referred for a diagnosis of tuberous sclerosis complex in the United States. \u003cem\u003eGenet. Med.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 88\u0026ndash;100 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones, A. C. et al. Comprehensive mutation analysis of TSC1 and TSC2-and phenotypic correlations in 150 families with tuberous sclerosis. \u003cem\u003eAm. J. Hum. Genet.\u003c/em\u003e \u003cb\u003e64\u003c/b\u003e, 1305\u0026ndash;1315 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosset, C. et al. TSC1 and TSC2 gene mutations and their implications for treatment in tuberous sclerosis complex: a review. \u003cem\u003eGenet. Mol. Biol.\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e, 69\u0026ndash;79 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKothare, S. V. et al. Severity of manifestations in tuberous sclerosis complex in relation to genotype. \u003cem\u003eEpilepsia\u003c/em\u003e \u003cb\u003e55\u003c/b\u003e, 1025\u0026ndash;1029 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi Napoli, C. et al. TSC1 and TSC2: tuberous sclerosis complex and its related epilepsy phenotype. \u003cem\u003eJ. Pediatr. Neurol.\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1055/s-0041-1727142\u003c/span\u003e\u003cspan address=\"10.1055/s-0041-1727142\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng, L-H. et al. Tsc2 gene inactivation causes a more severe epilepsy phenotype than Tsc1 inactivation in a mouse model of tuberous sclerosis complex. \u003cem\u003eHum. Mol. Genet.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, 445\u0026ndash;454 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNabbout, R., Kuchenbuch, M., Chiron, C. \u0026amp; Curatolo, P. Pharmacotherapy for Seizures in Tuberous Sclerosis Complex. \u003cem\u003eCNS Drugs\u003c/em\u003e. \u003cb\u003e35\u003c/b\u003e (9), 965\u0026ndash;983 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNabavi Nouri, M. et al. Epilepsy management in tuberous sclerosis complex: existing and evolving therapies and future considerations. \u003cem\u003ePediatr. Neurol.\u003c/em\u003e \u003cb\u003e126\u003c/b\u003e, 11\u0026ndash;19 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHenske, E. P. et al. Tuberous sclerosis complex. \u003cem\u003eNat. Rev. Dis. Primers\u003c/em\u003e. \u003cb\u003e2\u003c/b\u003e, 16035 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeliciano, D. M. et al. The neurodevelopmental pathogenesis of tuberous sclerosis complex (TSC). \u003cem\u003eFront. Neuroanat.\u003c/em\u003e ;\u003cb\u003e14\u003c/b\u003e. (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolton, P. F. et al. Intellectual abilities in tuberous sclerosis complex: risk factors and correlates from the Tuberous Sclerosis 2000 Study. \u003cem\u003ePsychol. Med.\u003c/em\u003e \u003cb\u003e45\u003c/b\u003e, 2321\u0026ndash;2331 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoinson, C. et al. Learning disability and epilepsy in an epidemiological sample of individuals with tuberous sclerosis complex. \u003cem\u003ePsychol. Med.\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e, 335\u0026ndash;344 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Vries, P. J. et al. Tuberous sclerosis complex-associated neuropsychiatric disorders (TAND): new findings on age, sex, and genotype in relation to intellectual phenotype. \u003cem\u003eFront. Neurol.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 603 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Eeghen, A. M. et al. Genotype and cognitive phenotype of patients with tuberous sclerosis complex. \u003cem\u003eEur. J. Hum. Genet.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, 510\u0026ndash;515 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEkong, R. et al. Variants within TSC2 exons 25 and 31 are very unlikely to cause clinically diagnosable tuberous sclerosis. \u003cem\u003eHum. Mutat.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e (4), 364\u0026ndash;370 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarach, L. S. et al. TSC2 c.1864C\u0026thinsp;\u0026gt;\u0026thinsp;T variant associated with mild cases of tuberous sclerosis complex. \u003cem\u003eAm. J. Med. Genet. A\u003c/em\u003e. \u003cb\u003e173\u003c/b\u003e (3), 771\u0026ndash;775 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJansen, A. C. et al. Unusually mild tuberous sclerosis phenotype is associated with TSC2 R905Q mutation. \u003cem\u003eAnn. Neurol.\u003c/em\u003e \u003cb\u003e60\u003c/b\u003e (5), 528\u0026ndash;539 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Eeghen, A. M. et al. Central TSC2 missense mutations are associated with a reduced risk of infantile spasms. \u003cem\u003eEpilepsy Res.\u003c/em\u003e \u003cb\u003e103\u003c/b\u003e (1), 83\u0026ndash;87 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;Connor, S. E. et al. A family with seizures and minor features of tuberous sclerosis and a novel TSC2 mutation. \u003cem\u003eNeurology\u003c/em\u003e \u003cb\u003e61\u003c/b\u003e (3), 409\u0026ndash;412 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWentink, M. et al. Functional characterization of the TSC2 c.3598C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.R1200W) missense mutation that co-segregates with tuberous sclerosis complex in mildly affected kindreds. \u003cem\u003eClin. Genet.\u003c/em\u003e \u003cb\u003e81\u003c/b\u003e (5), 453\u0026ndash;461 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhare, L. et al. A novel missense mutation in the GTPase activating protein homology region of TSC2 in two large families with tuberous sclerosis complex. \u003cem\u003eJ. Med. Genet.\u003c/em\u003e \u003cb\u003e38\u003c/b\u003e (5), 347\u0026ndash;349 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCamposano, S. et al. Distinct clinical characteristics of tuberous sclerosis complex patients with no mutation identified. \u003cem\u003eAnn. Hum. Genet.\u003c/em\u003e \u003cb\u003e73\u003c/b\u003e (2), 141\u0026ndash;146 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarach, L. S. et al. Drug-Resistant Epilepsy in Tuberous Sclerosis Complex Is Associated With TSC2 Genotype: More Findings From the Preventing Epilepsy Using Vigatrin (PREVeNT) Trial. \u003cem\u003ePediatr. Neurol.\u003c/em\u003e \u003cb\u003e159\u003c/b\u003e, 62\u0026ndash;71 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Ridder, J. et al. Early epileptiform EEG activity in infants with tuberous sclerosis complex predicts epilepsy and neurodevelopmental outcomes. \u003cem\u003eEpilepsia\u003c/em\u003e \u003cb\u003e62\u003c/b\u003e, 1208\u0026ndash;1219 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuratolo, P. et al. Advances in the genetics and neuropathology of tuberous sclerosis complex: edging closer to targeted therapy. \u003cem\u003eLancet Neurol.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 843\u0026ndash;856 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoavero, R. et al. Early clinical predictors of autism spectrum disorder in infants with tuberous sclerosis complex: results from the EPISTOP study. \u003cem\u003eJ. Clin. Med.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e (6), 788 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrueger, D. A. et al. Everolimus for treatment of tuberous sclerosis complex-associated neuropsychiatric disorders. \u003cem\u003eAnn. Clin. Transl Neurol.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e (12), 877\u0026ndash;887 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHwang, S-K. et al. Everolimus improves neuropsychiatric symptoms in a patient with tuberous sclerosis carrying a novel TSC2 mutation. \u003cem\u003eMol. Brain\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e, 56 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa\u0026aacute;sz, A. et al. Three-Year Follow-Up after Intrauterine mTOR Inhibitor Administration for Fetus with TSC-Associated Rhabdomyoma. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e (16), 12886 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamanta, D. Evolving treatment strategies for early-life seizures in Tuberous Sclerosis Complex: A review and treatment algorithm. \u003cem\u003eEpilepsy Behav.\u003c/em\u003e \u003cb\u003e161\u003c/b\u003e, 110123 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChu-Shore, C. J., Major, P., Camposano, S., Muzykewicz, D. \u0026amp; Thiele, E. A. The natural history of epilepsy in tuberous sclerosis complex. \u003cem\u003eEpilepsia\u003c/em\u003e \u003cb\u003e51\u003c/b\u003e (7), 1236\u0026ndash;1241 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoban, G. et al. Synthetic MRI in children with tuberous sclerosis complex. \u003cem\u003eInsights Imaging\u003c/em\u003e. \u003cb\u003e13\u003c/b\u003e (1), 115 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TSC1, TSC2, Tuberous Sclerosis Complex, Seizures, Genotype/Phenotype","lastPublishedDoi":"10.21203/rs.3.rs-5804009/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5804009/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTuberous Sclerosis Complex (TSC) is an autosomal dominant disorder characterized by widespread hamartomas in multiple organs and significant neurological involvement. TSC is caused by pathogenic variants in \u003cem\u003eTSC1\u003c/em\u003e or \u003cem\u003eTSC2\u003c/em\u003e genes, leading to hyperactivation of the mTOR pathway and consequent dysregulation of cell growth. These tumor suppressor genes encode hamartin and tuberin, proteins critical for regulating cell proliferation, neuronal excitability and synaptogenesis. In this retrospective study, we analyzed clinical, genetic and radiological features of 81 TSC patients from Sicily, focusing on genotype-phenotype correlations and intergroup comparisons. \u003cem\u003eTSC2\u003c/em\u003e mutations were more common than \u003cem\u003eTSC1\u003c/em\u003e mutations (61.7% vs. 38.3%). Patients with \u003cem\u003eTSC2\u003c/em\u003e mutations Patients with \u003cem\u003eTSC2\u003c/em\u003e mutations tended to exhibit a higher frequency of weekly seizures, a higher prevalence of infantile spasms and hypsarrhythmia compared to those with \u003cem\u003eTSC1\u003c/em\u003e mutations, consistent with a more severe phenotype. Interestingly, \u003cem\u003eTSC1\u003c/em\u003e patients exhibited a higher incidence of radial bands, while \u003cem\u003eTSC2\u003c/em\u003e patients harbored a larger average size of tubers and subependymal nodules. Cognitive and behavioral disorders were similarly distributed, although \u003cem\u003eTSC1\u003c/em\u003e patients had higher rates of normal or borderline cognitive function, while \u003cem\u003eTSC2\u003c/em\u003e patients had more severe neuropsychiatric profiles compared to \u003cem\u003eTSC1.\u003c/em\u003e Additionally, we present four novel potential genotype-phenotype correlations. To our knowledge, these is the first comprehensive \u003cem\u003eTSC1\u003c/em\u003e and \u003cem\u003eTSC2\u003c/em\u003e mutational analysis and genotype-phenotype correlation study carried out in in a large cohort of Sicilian patients affected by TSC. Our findings contribute to regional and global data on TSC, emphasizing the utility of genotype-informed management strategies.\u003c/p\u003e","manuscriptTitle":"Genetic Screening of Tuberous Sclerosis Complex in Sicily with a Focus on Neurological Manifestations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-14 23:02:20","doi":"10.21203/rs.3.rs-5804009/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-24T07:06:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-22T15:59:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-15T11:23:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49453877546801629858850486147236348829","date":"2025-04-12T14:17:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79940387379874465322883161147093284604","date":"2025-04-11T07:24:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-10T13:09:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-10T06:53:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-27T08:24:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fceb1e20-1ef0-4cdf-ba1f-d4e1c6dea587","owner":[],"postedDate":"April 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46976614,"name":"Health sciences/Neurology/Neurological disorders/Paediatric neurological disorders"},{"id":46976615,"name":"Biological sciences/Genetics/Clinical genetics"}],"tags":[],"updatedAt":"2025-06-30T15:58:49+00:00","versionOfRecord":{"articleIdentity":"rs-5804009","link":"https://doi.org/10.1038/s41598-025-04718-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-06-27 15:57:03","publishedOnDateReadable":"June 27th, 2025"},"versionCreatedAt":"2025-04-14 23:02:20","video":"","vorDoi":"10.1038/s41598-025-04718-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-04718-6","workflowStages":[]},"version":"v1","identity":"rs-5804009","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5804009","identity":"rs-5804009","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}