Paradigm shift in the treatment of tuberous sclerosis: Effectiveness of everolimus.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterised by abnormal cell proliferation and differentiation that affects multiple organs and can lead to the growth of hamartomas. Tuberous sclerosis complex is caused by the disinhibition of the protein mTOR (mammalian target of rapamycin). In the past, various therapeutic approaches, even if only symptomatic, have been attempted to improve the clinical effects of this disease. While all of these therapeutic strategies are useful and are still used and indicated, they are symptomatic therapies based on the individual symptoms of the disease and therefore not fully effective in modifying long-term outcomes. A new therapeutic approach is the introduction of allosteric inhibitors of mTORC1, which allow restoration of metabolic homeostasis in mutant cells, potentially eliminating most of the clinical manifestations associated with Tuberous sclerosis complex. Everolimus, a mammalian target of the rapamycin inhibitor, is able to reduce hamartomas, correcting the specific molecular defect that causes Tuberous sclerosis complex. In this review, we report the findings from the literature on the use of everolimus as an effective and safe drug in the treatment of TSC manifestations affecting various organs, from the central nervous system to the heart.

Menkes disease complicated by concurrent ACY1 deficiency: A case report.

Introduction: Menkes disease is an X-linked recessive condition caused by mutations in the ATP7A gene, which leads to severe copper deficiency. Aminoacylase-1 deficiency is a rare inborn error of metabolism caused by homozygous or compound heterozygous variant in the ACY1 gene, characterized by increased urinary excretion of specific N-acetyl amino acids. Case presentation: We report an infant with neurological findings such as seizures, neurodevelopmental delay and hypotonia. Metabolic screening showed low serum copper and ceruloplasmin, and increased urinary excretion of several N-acetylated amino acids. Whole-exome sequencing analysis (WES) revealed the novel de novo variant c.3642_3649dup (p.Ala1217Aspfs*2) in the ATP7A gene, leading to a diagnosis of Menkes disease, and the simultaneous presence of the homozygous ACY1 variant c.1057C>T (p.Arg353Cys) causative of Aminoacylase-1 deficiency. Conclusion: Our patient had two rare conditions with different treatment courses but overlapping clinical features. The identified novel ATP7A mutation associated with Menkes disease expands the ATP7A gene spectrum.

Rock around DYRK1A: Ethnic diversity, clinical challenges.

DYRK1A-related intellectual disability is a recently described syndrome characterized by microcephaly, global developmental delay, impaired speech development, and distinctive facial features, which let to define it as a recognizable syndrome. Here we report four new patients of different ethnicity, broadening the clinical phenotype of the condition and highlighting how ethnic influences in the facial appearance could make it less recognizable.

CGH Findings in Children with Complex and Essential Autistic Spectrum Disorder.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental condition with a strong genetic basis. We accurately assessed 209 ASD subjects, categorized in complex (47) and essential (162), and performed array comparative genomic hybridization to identify pathogenic and recurrent Copy Number Variants (CNVs). We found 117 CNVs in 75 patients, 11 classified as pathogenic. The complex ASD subjects have higher frequency of pathogenic CNVs with a diagnostic yield of 12.8%. Familiality, cognitive and verbal abilities, severity of autistic symptoms, neuroimaging and neurophysiological findings are not related to genetic data. This study identifies loci of interest for ASD and highlights the importance of a careful phenotypic characterization, as complex ASD is related to higher rate of pathogenic CNVs.

EEG at onset and MRI predict long-term clinical outcome in Aicardi syndrome.

OBJECTIVE: Descriptions of electroencephalographic (EEG) patterns in Aicardi syndrome (AIC) have to date referred to small cohorts of up to six cases and indicated severe derangement of electrical activity in all cases. The present study was conducted to describe the long-term EEG evolution in a larger AIC cohort, followed for up to 23 years, and identify possible early predictors of the clinical and EEG outcomes. METHODS: In a retrospective study, two experienced clinical neurophysiologists systematically reviewed all EEG traces recorded in 12 AIC cases throughout their follow-up, from epilepsy onset to the present. Clinical outcome was assessed with standardized clinical outcome scales. RESULTS: Analysis of the data revealed two distinct AIC phenotypes. In addition to the "classical severe phenotype" already described in the literature, we identified a new "mild phenotype". The two phenotypes show completely different EEG features at onset of epilepsy and during its evolution, which correspond to different clinical outcomes. CONCLUSIONS: Data from our long-term EEG and clinical-neuroradiological study allowed us to describe two different phenotypes of AIC, with different imaging severity and, in particular, different EEG at onset, which tend to remain constant over time. SIGNIFICANCE: Together, these findings might help to predict long-term clinical outcomes.

Cognitive, Behavioral and Socioemotional Development in a Cohort of Preterm Infants at School Age: A Cross-Sectional Study.

More than 50% of children who survive prematurity have an atypical course of development at school age, as environmental demands become more demanding. This study examines the effects of preterm birth on the cognitive, behavioral and socioemotional development of 185 children at ages five and seven years. Weaknesses were found in attention, working memory, processing speed and the ability to correctly interpret emotions at both ages five and seven. Significant correlations were found in regression and moderation models. These findings suggest that school-age children who were preterm infants are at increased risk of exhibiting impairments in several developmental domains that may affect their overall quality of life.

Progressive Clinical and Neuroradiological Findings in a Child with BCL11B Missense Mutation: Expanding the Phenotypic Spectrum of Related Disorder.

We report a patient affected by BCL11B-related disorder, providing the first extensive demonstration of clinical and neuroradiological progressive course of the disease, with possible implications on the way it is studied and followed-up. Never described clinical aspects such as toes abnormalities and hypospadias widen the range of dysmorphisms associated with this condition. Our data suggest that BCL11B mutations may be implicated not only in impaired morphogenesis and hematopoiesis but also in progressive central nervous system damage, which remains to be further investigated and clarified.

Prenatal Diagnosis and Neurodevelopmental Outcome in Isolated Cerebellar Hypoplasia of Suspected Hemorrhagic Etiology: a Retrospective Cohort Study.

Data about the neurological prognosis of isolated cerebellar hypoplasia in utero are scant and inconsistent. In this monocentric retrospective study, we describe the neurodevelopmental outcomes in a series of children with isolated cerebellar hypoplasia of presumably hemorrhagic origin prenatally detected with fetal magnetic resonance imaging (fMRI). We retrospectively reviewed the charts of all the pregnant women who were referred for a neurological consultation, diagnosed with fetal encephalic malformation/disruption between 2010 and 2020 in the Fetal Therapy Unit of our institution. Fetal MRI (fMRI) was performed in all the pregnancies. Fetuses with cerebellar hypoplasia presumably of hemorrhagic origin were selected for the study. Fetuses exposed to alcohol or with additional malformations in other cerebral or body areas were excluded. All the infants received the postpartum follow-up care adopted in our center, including post-natal MRI, serial neurological examinations, standardized neurodevelopmental tests, and regular parental interviews. Cognitive functions were tested with GRIFFITHS II, WPPSI-III, and WISC-IV according to the child's age. A total of 14 pregnant women out of 479 fetal consultations were eligible and included in the study group. In 57% of cases, the etiology of the hemorrhage was unknown. In 21% of cases, it was attributed to a blood transfusion, while in the remaining ones, it was attributed to maternal predisposing factors. Among the survivors, two infants were excluded for prematurity, and two were lost to follow-up. Ten patients were thus included in the study. Six patients had normal neurodevelopment and cognition, and three presented mild-moderate neurological signs, i.e., mild dyspraxia and visuoperceptual impairment. Only one child had a severe outcome, i.e., autism spectrum disorder. The cerebellum is particularly vulnerable to disruptions throughout its prolonged development. Extreme caution must be used in prenatal counseling considering that in the acute phase, lesion extension and vermis involvement can be overestimated with fMRI. In cases of uncertainty, performing an additional fMRI could be advisable after 4-8 weeks. However, in our series, infants with isolated cerebellar hypoplasia tended to have a favorable prognosis. Nevertheless, a long-term follow-up is needed and should include a postnatal brain MRI, serial neurological examinations, and neurodevelopmental tests at least up to school age.

Etiological research in pediatric multiple sclerosis: A tool to assess environmental exposures (PEDiatric Italian Genetic and enviRonment ExposurE Questionnaire).

BACKGROUND: The etiology of pediatric-onset multiple sclerosis is unknown although putative genetic and environmental factors appear to be involved. Among children multiple sclerosis onset occurs closer to the susceptibility window thank in adults and the exposure to etiological environmental factors is more informative. An Italian multicentre case-control study (the PEDiatric Italian Genetic and enviRonment ExposurE, PEDIGREE study) was designed to investigate environmental exposures in pediatric-onset multiple sclerosis and their interaction with genetics. OBJECTIVES: To collect evidence on exposures to environmental risk factors in pediatric-onset multiple sclerosis, a questionnaire was developed for the Italian population (PEDIGREE Questionnaire) and is presented. METHODS: PEDIGREE Questionnaire develops from an existing tool used in case-control studies on pediatric-onset multiple sclerosis in US Americans, and was translated, adapted and tested for the contents perceived relevance, acceptability, feasibility and reliability in a population of Italian pediatric subjects and their parents recruited from clinics and general population. RESULTS: PEDIGREE Questionnaire contents were overall deemed relevant by the study population, acceptable for 100% participants and feasible for at least 98%. PEDIGREE Questionnaire degree of reliability ranged 56% to 72%. CONCLUSION: PEDIGREE Questionnaire proves to be an efficient tool to assess environmental exposures in the Italian pediatric population. We encourage the dissemination of population-specific questionnaires and shared methodology to optimize efforts in MS etiological research.

CDKL5 deficiency disorder in males: Five new variants and review of the literature.

The X-linked Cyclin-Dependent Kinase-Like 5 (CDKL5) gene encodes a serine-threonine kinase highly expressed in the developing brain. Loss of function of CDKL5 is pointed out to underlie the CDKL5 Deficiency Disorder (CDD), an X-linked dominant disease characterized by early-onset epileptic encephalopathy and developmental delay, usually affecting females more than males. To the best to our knowledge, only 45 males with CDD have been reported so far. Type and position of CDKL5 variants with different impact on the protein are reported to influence the clinical presentation. X-chromosome inactivation occurring in females and post-zygotic mosaicism in males are also believed to contribute to this variability. Based on these issues, genotype-phenotype correlations are still challenging. Here, we describe clinical features of five additional affected males with unreported CDKL5 variants, expanding the molecular spectrum of the disorder. We also reviewed the clinical profile of the previously reported 45 males with molecularly confirmed CDD. Severe developmental delay, cortical visual impairment, and early-onset refractory epilepsy characterize the CDD picture in males. By assessing the molecular spectrum, we confirm that germ-line truncating CDKL5 variants, equally distributed across the coding sequence, are the most recurrent mutations in CDD, and cause the worsen phenotype. While recurrence and relevance of missense substitutions within C-terminal remain still debated, disease-causing missense changes affecting the N-terminal catalytic domain correlate to a severe clinical phenotype. Finally, our data provide evidence that post-zygotic CDKL5 mosaicism may result in milder phenotypes and, at least in a subset of subjects, in variable response to antiepileptic treatments.

Efficacy of Everolimus Low-Dose Treatment for Cardiac Rhabdomyomas in Neonatal Tuberous Sclerosis: Case Report and Literature Review.

Background: Cardiac rhabdomyomas (CRs) are the most common cardiac tumors in newborns. Approximately 80-90% of cases are associated with tuberous sclerosis complex (TSC). In selective cases, Everolimus has resulted in a remarkable tumoral regression effect in children with TS. The optimal dosage for neonates is still unknown. Case presentation: We describe the use of Everolimus in a neonate with multiple biventricular CRs, causing subaortic obstruction, in which a low-dose treatment (0.1 mg/die), in an effort to maintain serum trough levels of 3-7 ng/mL, was successfully used off-label, without adverse effects. Conclusions: We showed that a low-dose Everolimus regimen may be an effective and safe treatment for CR regression in TS neonates, when the minimum therapeutic range was maintained.

Neurological phenotype of Potocki-Lupski syndrome.

Potocki-Lupski syndrome is a condition mainly characterized by infantile hypotonia, developmental delay/intellectual disability (DD/ID), and congenital anomalies, caused by duplications of the 17p11.2 region, encompassing RAI1 gene. Its clinical presentation is extremely variable, especially for what concerns the cognitive level and the behavioral phenotype. Such aspects, as well as the dysmorphic/malformative ones, have been covered by previous studies; otherwise neurological features have never been systematically described. In order to delineate the neurological phenotype of Potocki-Lupski Syndrome, we collect an 8-patients cohort. Developmental milestones are delayed and a mild to moderate cognitive impairment is present in all patients, variably associated with features of autism spectrum disorder, behavioral disturb, and sleep disturb. Hypotonia appears a less frequent finding than what previously reported, while motor clumsiness/coordination impairment is frequent. EGG registration demonstrated a common pattern with excess of diffuse rhythmic activity in sleep phases or while the patient is falling asleep. Brain MRI did not reveal common anomalies, although unspecific white matter changes may be present. We discuss such findings and compare them to literature data, offering an overview on the neurological and cognitive-behavioral presentation of the syndrome.

Clinical spectrum of PTEN mutation in pediatric patients. A bicenter experience.

OBJECTIVE OF THE STUDY: To give a full overview of the clinical presentation of PTEN mutations in pediatric patients and to propose a pediatric follow-up protocol. METHODS: Recruitment of 16 PTEN mutated children (age 6 months-11 years) from two pediatric centers in Milan (Italy) between 2006 and 2017. All the patients underwent clinical and neurologic evaluations, cognitive and behavioral tests, and brain MRI; they are currently following an oncologic follow-up. RESULTS: Extreme macrocephaly is present in all the patients (69% HC above +4 SD). Neuropsychiatric issues have high prevalence, with 56% of patients showing developmental delay and 25% showing autism spectrum disorder. Brain MRI reveals in 75% of the patients at least one of the following: enlarged perivascular spaces, white matter anomalies, and/or downward displacement of the cerebellar tonsils through the foramen magnum, resulting in Chiari I malformation in two patients. Vascular malformations have a prevalence of 19%, with further evidence that complex cardiovascular malformations may be related to PTEN mutations; 31% of patients present hamartomas. None of our patients have so far experienced any oncologic complication. CONCLUSIONS: We suggest to screen for PTEN mutations all children presenting macrocephaly and one of the following: neurodevelopmental issues, one of the three major brain MRI anomalies, cutaneous lesions, vascular malformations, family history positive for PTEN related malignancies; or also with macrocephaly alone when exceeding +3 SD. Basing on our cohort results and further recent studies on the condition, we recommend a follow-up protocol that includes annual clinical and dermatological examination, thyroid and abdominal US, and Fecal Occult Blood test plus neurodevelopmental evaluation, heart US (to exclude congenital heart malformations), and brain MRI (to exclude Chiari I malformation) at diagnosis.

Concurrent AFG3L2 and SPG7 mutations associated with syndromic parkinsonism and optic atrophy with aberrant OPA1 processing and mitochondrial network fragmentation.

Mitochondrial dynamics and quality control are crucial for neuronal survival and their perturbation is a major cause of neurodegeneration. m-AAA complex is an ATP-dependent metalloprotease located in the inner mitochondrial membrane and involved in protein quality control. Mutations in the m-AAA subunits AFG3L2 and paraplegin are associated with autosomal dominant spinocerebellar ataxia (SCA28) and autosomal recessive hereditary spastic paraplegia (SPG7), respectively. We report a novel m-AAA-associated phenotype characterized by early-onset optic atrophy with spastic ataxia and L-dopa-responsive parkinsonism. The proband carried a de novo AFG3L2 heterozygous mutation (p.R468C) along with a heterozygous maternally inherited intragenic deletion of SPG7. Functional analysis in yeast demonstrated the pathogenic role of AFG3L2 p.R468C mutation shedding light on its pathogenic mechanism. Analysis of patient's fibroblasts showed an abnormal processing pattern of OPA1, a dynamin-related protein essential for mitochondrial fusion and responsible for most cases of hereditary optic atrophy. Consistently, assessment of mitochondrial morphology revealed a severe fragmentation of the mitochondrial network, not observed in SCA28 and SPG7 patients' cells. This case suggests that coincidental mutations in both components of the mitochondrial m-AAA protease may result in a complex phenotype and reveals a crucial role for OPA1 processing in the pathogenesis of neurodegenerative disease caused by m-AAA defects.

The noncoding RNA AK127244 in 2p16.3 locus: A new susceptibility region for neuropsychiatric disorders.

The presence of redundant copy number variants (CNVs) in groups of patients with neurological diseases suggests that these variants could have pathogenic effect. We have collected array comparative genomic hybridization (CGH) data of about 2,500 patients affected by neurocognitive disorders and we observed that CNVs in 2p16.3 locus were as frequent as those in 15q11.2, being both the most frequent unbalances in our cohort of patients. Focusing to 2p16.3 region, unbalances involving NRXN1 coding region have been already associated with neuropsychiatric disorders, although with incomplete penetrance, but little is known about CNVs located proximal to the gene, in the long noncoding RNA AK127244. We found that, in our cohort of patients with neuropsychiatric disorders, the frequency of CNVs involving AK127244 was comparable to that of NRXN1 gene. Patients carrying 2p16.3 unbalances shared some common clinical characteristics regardless NRXN1 and AK127244 CNVs localization, suggesting that the AK127244 long noncoding RNA could be involved in neurocognitive disease with the same effect of NRXN1 unbalances. AK127244 as well as NRXN1 unbalances seem to have a particular influence on language development, behavior or mood, according with the topographic correlation between NRXN1 expression and prefrontal cortex functions.

Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly.

Mutations of genes within the phosphatidylinositol-3-kinase (PI3K)-AKT-MTOR pathway are well known causes of brain overgrowth (megalencephaly) as well as segmental cortical dysplasia (such as hemimegalencephaly, focal cortical dysplasia and polymicrogyria). Mutations of the AKT3 gene have been reported in a few individuals with brain malformations, to date. Therefore, our understanding regarding the clinical and molecular spectrum associated with mutations of this critical gene is limited, with no clear genotype-phenotype correlations. We sought to further delineate this spectrum, study levels of mosaicism and identify genotype-phenotype correlations of AKT3-related disorders. We performed targeted sequencing of AKT3 on individuals with these phenotypes by molecular inversion probes and/or Sanger sequencing to determine the type and level of mosaicism of mutations. We analysed all clinical and brain imaging data of mutation-positive individuals including neuropathological analysis in one instance. We performed ex vivo kinase assays on AKT3 engineered with the patient mutations and examined the phospholipid binding profile of pleckstrin homology domain localizing mutations. We identified 14 new individuals with AKT3 mutations with several phenotypes dependent on the type of mutation and level of mosaicism. Our comprehensive clinical characterization, and review of all previously published patients, broadly segregates individuals with AKT3 mutations into two groups: patients with highly asymmetric cortical dysplasia caused by the common p.E17K mutation, and patients with constitutional AKT3 mutations exhibiting more variable phenotypes including bilateral cortical malformations, polymicrogyria, periventricular nodular heterotopia and diffuse megalencephaly without cortical dysplasia. All mutations increased kinase activity, and pleckstrin homology domain mutants exhibited enhanced phospholipid binding. Overall, our study shows that activating mutations of the critical AKT3 gene are associated with a wide spectrum of brain involvement ranging from focal or segmental brain malformations (such as hemimegalencephaly and polymicrogyria) predominantly due to mosaic AKT3 mutations, to diffuse bilateral cortical malformations, megalencephaly and heterotopia due to constitutional AKT3 mutations. We also provide the first detailed neuropathological examination of a child with extreme megalencephaly due to a constitutional AKT3 mutation. This child has one of the largest documented paediatric brain sizes, to our knowledge. Finally, our data show that constitutional AKT3 mutations are associated with megalencephaly, with or without autism, similar to PTEN-related disorders. Recognition of this broad clinical and molecular spectrum of AKT3 mutations is important for providing early diagnosis and appropriate management of affected individuals, and will facilitate targeted design of future human clinical trials using PI3K-AKT pathway inhibitors.

ZC4H2 deletions can cause severe phenotype in female carriers.

ZC4H2 is involved in human brain development, and, if mutated, can be responsible for a rare X-linked disorder, originally presented in literature as Wieacker-Wolff syndrome and Miles-Carpenter syndrome. In males, severe intellectual disability is associated with variable symptoms of central and peripheral nervous system involvement, such as spasticity, hyperreflexia, muscle weakness, and arthrogryposis. Female carriers are usually described as asymptomatic or only mildly affected. Here, we report on a girl carrying a de novo deletion of ZC4H2 detected by array-CGH analysis. She showed a complex neurodevelopmental disorder resembling the clinical picture commonly observed in male patients. X-inactivation was found to be random. Additionally, she had an unusual appearance of fingers and hand creases, and electromyography showed a peculiar pattern of both neurogenic and myopathic anomalies. The present patient confirms that female carriers can also be severely affected. Systematic clinical investigations of both males and females are needed to define the variety in nature and severity of phenotypes related to ZC4H2 variants.

Insights into 6q21-q22: Refinement of the critical region for acro-cardio-facial syndrome.

Deletions on chromosome 6q are rarely reported in the literature, and genotype-phenotype correlations are poorly understood. We report a child with a deletion of the 6q21-q22 chromosomal region, providing some intriguing results about the correlation between this region and acro-cardio-facial syndrome, congenital heart disease, split hand and foot malformation, and epilepsy.

Chromosome 17q21.31 duplication syndrome: Description of a new familiar case and further delineation of the clinical spectrum.

INTRODUCTION: 17q21.31 microduplication syndrome is a recently described condition associated with a broad clinical spectrum, of which psychomotor delay, behavioral disorders and poor social interaction seem to be the most consistent features. Only seven patients have been reported thus far. All have behavioral disorders reminiscent of the autistic spectrum with intellectual skills ranging from normal to mild intellectual deficiency. Other features are variable with no striking common phenotypic features. CASE STUDY: Here we describe the segregation of 17q21.31 duplication in an Italian family. DISCUSSION: Clinical features and genetic data are reported, and compared with previously reported patients with 17q21.31 microduplication. A comparison of clinical manifestations between deletion and duplication syndromes of the chromosome regione is provided.

The Diagnostic Yield of Array Comparative Genomic Hybridization Is High Regardless of Severity of Intellectual Disability/Developmental Delay in Children.

Microarray-based comparative genomic hybridization is a method of molecular analysis that identifies chromosomal anomalies (or copy number variants) that correlate with clinical phenotypes. The aim of the present study was to apply a clinical score previously designated by de Vries to 329 patients with intellectual disability/developmental disorder (intellectual disability/developmental delay) referred to our tertiary center and to see whether the clinical factors are associated with a positive outcome of aCGH analyses. Another goal was to test the association between a positive microarray-based comparative genomic hybridization result and the severity of intellectual disability/developmental delay. Microarray-based comparative genomic hybridization identified structural chromosomal alterations responsible for the intellectual disability/developmental delay phenotype in 16% of our sample. Our study showed that causative copy number variants are frequently found even in cases of mild intellectual disability (30.77%). We want to emphasize the need to conduct microarray-based comparative genomic hybridization on all individuals with intellectual disability/developmental delay, regardless of the severity, because the degree of intellectual disability/developmental delay does not predict the diagnostic yield of microarray-based comparative genomic hybridization.