Rare diseases (RDs), affecting fewer than 5 people per 10,000, present unique challenges to usual care pathways due to their unique characteristics: rarity and large number of disease entities, heterogeneous clinical manifestations and genetic causes, multisystemic involvement, and high complexity of diagnosis and treatment. This complexity often hampers the setting of appropriate pathways of care, which are not easily identifiable by patients and stakeholders. This ultimately leads to significant delays in diagnosis, lack of timely access to RD treatments and profound inequalities across countries. To overcome these difficulties, European Reference Networks (ERNs) were established in 2017 to facilitate patients' referral to expertise and excellent services, aiming to reduce disparities and expedite diagnosis, standard of care, and treatment for people living with rare diseases (PLWRDs). Since 72% of rare diseases are of genetic in origin and mostly affect children, genomic newborn screening (gNBS) offers a powerful tool to overcome diagnostic barriers by providing early and accurate genetic diagnoses for a wide range of treatable pediatric RDs. Several gNBS initiatives have been implemented across Europe and worldwide. Screen4Care (S4C) is an EU-IHI funded research project integrating gNBS with artificial intelligence (AI)-based tools to improve care for PLWRDs in the EU. The project will offer gNBS to up to 18,000 infants using a capture-based panel (TREAT-panel) targeting 245 genes associated with treatable genetic disorders [ClinicalTrials.gov NCT06549218]. Within this framework, an operational pipeline and a comprehensive step-by-step process in collaboration with ERNs were developed to refer gNBS-positive newborns to the appropriate ERN, ensuring timely access to optimal standards of care and available treatments. We suggest that this organisational and structured health model might be adopted by EU Member States (MS), as it provides a defined clinical framework for identifying newborns with RDs at birth and ensuring they receive the correct care, thereby promoting patient-centred and equitable disease management.

Newborn screening (NBS) and its genetic version, genetic NBS (gNBS), are now used to identify a broad range of conditions, including metabolic, endocrine, and genetic disorders, leading to significant reductions in infant mortality and long-term complications. Advances in genomic technologies, particularly next-generation sequencing, have enhanced the ability to detect rare diseases early, using gNBS, improving long-term outcomes. The availability and scope of gNBS vary across countries, influenced by national policies and technological advancements. This systematic literature review aims to clarify the specific barriers, opportunities, and more general attitudes that stakeholders express about gNBS for rare diseases. We extracted articles from 2010 to 2022. We followed the PRISMA guidelines and registered the review via PROSPERO (CRD42022297678). From an initial retrieval of 4519 records, two selection rounds resulted in a final list of 112 articles, which were assessed across different categories exploring various aspects of gNBS. The most important perceived opportunities in gNBS were the benefits of early intervention to reduce the burden of the diagnostic odyssey. The main identified barriers included three key codes: the stress and risk associated with false results and dealing with uncertainty (n = 25), the psychosocial implications (n = 26), and misunderstandings due to lack of education or communication. The majority of respondents expressed positive views, particularly regarding actionability. The results indicate a generally favourable attitude toward newborn screening, with subtle variations in viewpoints. Our findings on these themes can specifically inform how final attitudes are shaped based on particular aspects.

This study aimed to describe and molecularly characterize a rare case of early-onset multiple primary tumors associated with a novel germline protection of telomeres 1 (POT1) gene pathogenic variant, associated with POT1 tumor predisposition syndrome (POT1-TPD), a rare autosomal dominant disorder characterized by an increased risk for various tumors, including gliomas. A custom clinical exome panel for genes associated with Cancer Predisposition Syndromes was performed on DNA extracted from blood. RNA sequencing and genome-wide DNA methylation profiling were conducted on tumors. We report the case of a female patient diagnosed at age 12 with a diffuse glioma harboring a ROS1 fusion. At age 18, she developed a renal cell carcinoma. Genetic germline testing revealed a heterozygous germline POT1 variant, c.910dupG (p.Asp304fs*8), classified as pathogenic. Segregation analysis demonstrated paternal inheritance of the variant. The same POT1 variant was identified in both tumor tissues in a heterozygous state. This is the first reported case of a young adult carrying a new pathogenic POT1 variant who developed a pediatric high-grade glioma and a renal tumor in early adulthood. This report expands the clinical spectrum of POT1-associated tumors with early-onset and underscores the relevance of genetic testing for patient management and family counseling.

The relationship between pediatric and adolescent/young adult (AYA) patients with high-grade gliomas (pediatric high-grade gliomas [pHGGs]) and cancer predisposition syndromes (CPSs) remains insufficiently explored, despite the increasing use of massive parallel sequencing in the diagnostic setting. We retrospectively analyzed sequencing data from 95 pediatric patients diagnosed with HGGs to investigate the presence of germline variants associated with cancer risk. The presence of somatic variants was also evaluated in 15 affected individuals. In silico and in vitro studies were performed to reclassify one variant of uncertain significance (VUS). We identified 80 variants across the 95 patients, including 17 pathogenic (P), 2 likely pathogenic (LP), 60 VUSs, and 1 likely benign (LB), after reclassification. Notably, 23.7% of the P/LP variants were found in genes associated with CPSs. While the distribution of these variants did not show significant differences across tumor subtypes, the highest proportion of P/LP variants was observed in diffuse midline gliomas. Functional studies led to the reclassification of one LZTR1 variant from a VUS to LP. The collected data revealed that 18.9% of patients had P/LP variants; notably, among the 11.6% of patients carrying P/LP variants, there were variants in genes known to be associated with the development of central nervous system (CNS) tumors in pediatric and AYA patients, a rate higher than the 10% incidence typically reported in the literature for pediatric CNS tumors. This finding underscores the value of our comprehensive analysis for germline variants in HGG, suggesting a greater prevalence of CPS in these patients than previously reported.

Abstract Background Germline pathogenic and likely pathogenic variants (PV/LPV) in MutY DNA Glycosylase (MUTYH), a key gene in the Base Excision Repair (BER) pathway, are classically linked to MUTYH-associated polyposis (MAP) and colorectal cancer. Emerging evidence suggests that MUTYH variants, particularly monoallelic ones, may contribute to susceptibility to other malignancies, including central nervous system (CNS) tumors. Methods We retrospectively analyzed 378 pediatric and adolescent/young adult (AYA) patients (222 males, 156 females) with CNS tumors diagnosed between 2011 and 2025. All patients underwent clinical exome sequencing, which included analysis of the MUTYH gene. Tumors were classified according to the WHO CNS 5th edition. Variant inheritance was determined by trio-based next-generation sequencing (NGS) and classified according to ACMG guidelines. Results MUTYH variants were found in 15/378 patients: PV/LPV were detected in 11 patients (2.9%) and variants of uncertain significance (VUSs) in 4 (1.1%). One patient carried biallelic variants p.Glu452del and p.Tyr76*; while ten patients had monoallelic variants: p.Tyr179Cys (n = 4), p.Gly368Asp (n = 5) and p.Arg242His (n = 1). The prevalence of the rs34612342 and rs36053993 variants was 1.1% and 1.3%, respectively. The Sixty percent of cases with a MUTYH variant reported a positive family history for cancer. In this large single-institution cohort of pediatric and AYA patients with a diagnosis of a CNS tumor, 4.0% of patients carried germline MUTYH variants, predominantly monoallelic pathogenic or likely pathogenic substitutions. Conclusions Our findings support the inclusion of MUTYH in multigene germline testing panels for pediatric CNS tumors and warrant further investigation into its mechanistic and clinical significance as a low-penetrance susceptibility factor in neuro-oncogenesis.

Multiple endocrine neoplasia type 2B (MEN2B) is a rare autosomal-dominant cancer predisposition syndrome in which prognosis is strongly influenced by the stage of medullary thyroid carcinoma at diagnosis. Virtually all affected children exhibit 1 or more extra-endocrine features during childhood, offering an opportunity for timely recognition. We retrospectively reviewed 5 pediatric patients with MEN2B managed at Bambino Gesù Children’s Hospital, all carrying the highest-risk RET proto-oncogene mutation (M918T); 4 cases were de novo and 1 was maternally inherited. In each case, genetic testing was prompted by extra-endocrine manifestations observed during infancy or early childhood, including chronic constipation, congenital clubfoot, and alacrimia. Recognition of these early clinical features led to genetic diagnosis before or at the onset of endocrine disease and enabled timely surgical management. This case series highlights the spectrum of extra-endocrine manifestations that may precede endocrine disease in MEN2B and emphasizes their value as early diagnostic clues. Early recognition of these features may facilitate prompt genetic testing and timely thyroidectomy before metastatic medullary thyroid carcinoma develops.

Endolysosomal abnormalities are particularly detrimental to the nervous system and have been implicated in neuropsychiatric disorders. Key regulators of the lysosomal and endosomal luminal ion homeostasis are CLC chloride/proton exchangers. We report 15 individuals carrying variants in CLCN3, encoding a ubiquitous endosomal 2Cl−/H+ exchanger, and provide updated clinical information for 5 previously reported individuals. Subjects displayed a broad spectrum of neuropsychiatric symptoms, including developmental delay, intellectual disability, and epilepsy. To reveal the pathogenic mechanism, we investigated ClC-3 variants-mediated ion transport and its regulation by the recently discovered inhibitory beta subunit TMEM9. 12/20 missense variants exhibited altered properties and fell into two classes: those affecting the region binding inhibitory TMEM9 carboxy-termini, and those that broaden the voltage range over which ClC-3 conducts ions. Surprisingly, the latter variants also attenuated TMEM9-mediated inhibition. Both classes produced a toxic gain-of-function, as evident from endolysosomal vacuolization by mutant ClC-3/TMEM9 overexpression. Our results expand the genetic and clinical spectrum of CLCN3-related disease, provide a solid basis for genetic counseling, and uncover an unexpected link between gating-associated conformational changes and inhibition by TMEM9. Loss- and gain-of-function variants of the endosomal chloride/proton exchanger ClC-3 are associated with neurodevelopmental disorders. Identification and characterization of novel variants expands the clinical spectrum of CLCN3 disease and provides detailed insights into pathogenic mechanisms. Loss- and gain-of-function variants of the endosomal chloride/proton exchanger ClC-3 are associated with neurodevelopmental disorders. Identification and characterization of novel variants expands the clinical spectrum of CLCN3 disease and provides detailed insights into pathogenic mechanisms.

Abstract Background BRCA1/BRCA2 germline variants are well-established cancer predisposition factors in adults, yet their contribution to central nervous system (CNS) tumor susceptibility in pediatric and adolescent/young adult (AYA) patients remains uncertain. Recent pediatric sequencing studies suggest that heterozygous BRCA2 pathogenic variants may act as low-penetrance susceptibility alleles in selected CNS tumors. Methods We retrospectively reviewed 367 pediatric and AYA patients with primary CNS tumors diagnosed between 2011 and 2025 at our center, all of whom underwent clinical exome sequencing including BRCA1 and BRCA2. Variants were classified per ACMG criteria. Segregation was assessed by trio testing when available or by single-parent testing. Somatic sequencing was performed in a subset of cases. Results Germline BRCA1/2 variants were identified in 17/367 patients (4.6%). Pathogenic/likely pathogenic (P/LP) variants were detected in 5/17 (29%): four monoallelic truncating pathogenic variants in BRCA2 and one monoallelic likely pathogenic variant in BRCA1. The remaining 12/17 (71%) carried variants of uncertain significance (VUS), including two BRCA1 VUS and multiple BRCA2 VUS, with two patients harboring more than one VUS. Variant-positive cases spanned multiple histologies, including high-grade glioma, low-grade glioma, and medulloblastoma, supporting a non–tumor-type–restricted distribution. Family history of cancer was positive in 8/17 (47.1%). Inheritance assessment was available for most patients (15/17): 64.7% showed maternal inheritance. Tumor sequencing was limited and did not allow systematic evaluation of biallelic inactivation, loss of heterozygosity, or homologous recombination deficiency. Conclusions In this large single-center pediatric/AYA CNS tumor cohort, monoallelic BRCA1/2 P/LP variants were uncommon but detectable, with a predominance of truncating BRCA2 alterations across diverse CNS tumor entities. These data are consistent with a model in which heterozygous BRCA variants may function as low-penetrance susceptibility factors rather than primary drivers of tumorigenesis. Inclusion of BRCA1/2 in germline testing panels may inform genetic counseling, cascade testing, and long-term surveillance.

Loss-of-function variants in the WW Domain Containing Adaptor with Coiled-Coil (WAC) gene are associated with DeSanto-Shinawi syndrome (DESSH), a rare autosomal dominant neurodevelopmental disorder usually with onset characterized by global developmental delay appearing in infancy or early youth, intellectual disability, seizures, autism spectrum disorder, attention-deficit/hyperactivity disorder, hypotonia, dysmorphic features at the level of the skull and face. The protein encoded by the WAC gene links and regulates gene transcription and monoubiquitination of histone H2B to "Lys-120" (H2BK120ub1). It also acts in the regulation of cell cycle checkpoint activation in response to DNA damage, and the RING finger 20/40 (RNF20/40)/WAC complex has been proven to act as an interactor with p53. We describe a 15-year-old boy with a diagnosis of DESSH associated with a WAC variant and a polymorphous low-grade neuroepithelial tumor of the young associated with an FGFR2(ex17):INA(ex2) fusion. In addition, two germinal likely pathogenic variants have been identified in the Neurofibromin 1 (NF1) and succinate dehydrogenase complex flavoprotein subunit A (SDHA) genes. Our data suggest a possible additional role of the WAC variant in early tumor development, highlighting the importance of oncogenetic testing in patients with rare neurodevelopmental syndromes and brain tumors.

Tubulinopathies constitute a growing and heterogeneous class of disorders caused by pathogenic variants in genes encoding for tubulins, the structural components of microtubules. These conditions encompass a broad spectrum of neurological manifestations, most commonly including structural brain malformations and, less frequently, neurodegenerative disorders. In addition to brain diseases, tubulinopathies also include non-neurological conditions, such as female meiotic infertility and bleeding disorders. Among tubulin genes, TUBB, encoding the class I β-tubulin, has been associated with a wide range of clinical conditions, primarily including neurodevelopmental delay, structural brain abnormalities, and skin creases. More recently, TUBB has also been linked to inherited macrothrombocytopaenia, suggesting a broader, yet still incompletely defined, role of this β-tubulin in haematopoiesis and platelet biogenesis. In this study, we aim to investigate pathogenic mechanisms of a novel frameshift variant in TUBB (c.628dup; p.Ile210AsnfsTer40), identified in a subject affected by thrombocytopaenia and neurological involvement. Functional analyses demonstrated that the p.Ile210AsnfsTer40 TUBB mutation leads to the production of a truncated β-tubulin with reduced protein stability and aberrant subcellular localization. The mutant protein showed impaired interaction with α-tubulin and kinesin family member 1 A (KIF1A), suggesting defective microtubule assembly and microtubule-mediated intracellular transport. Furthermore, the inhibition of the proteasome degradative pathway resulted in a marked increase of the mutant TUBB, indicating altered proteostasis and enhanced degradation of the truncated tubulin. Finally, we showed that the p.Asp249Val substitution in TUBB, previously associated with macrothrombocytopaenia, similarly reduces protein stability, weakens interaction with α-tubulin, and impairs TUBB incorporation into microtubules. This study associates the novel heterozygous truncating p.Ile210AsnfsTer40 variant in TUBB with a multisystem disorder characterized by haematological and neurological manifestations, expanding the phenotypic spectrum of TUBB-related diseases. Our findings indicate that reduced protein stability and functional impairment of β-tubulin represent key pathogenic mechanisms underlying this condition. Lastly, our experimental evidence further supports the implication of TUBB dysfunctions in haematological abnormalities, offering additional insight into the molecular basis and clinical variability of tubulin-related disorders.

Abstract Background Defects in the DNA damage response, particularly within the homologous recombination (HR) pathway, are increasingly recognized as drivers of cancer predisposition. Members of the RAD gene family are central to HR-mediated DNA repair, yet their role in pediatric central nervous system (CNS) tumor susceptibility remains poorly characterized. Methods We performed a descriptive analysis of 84 pediatric patients with CNS tumors who underwent germline testing including RAD family genes. Clinical, histopathological, and family history data were collected. Variants were classified according to ACMG criteria. Trio analysis was performed when available, and tumor tissue sequencing was conducted in selected cases. Results Thirteen patients (15.5%) harbored germline variants in RAD genes. The most frequently involved genes were RAD54L (35.7%), RAD51D (28.7%), RAD50 (21.4%), RAD51B (7.1%), and RAD52 (7.1%). Diagnoses included high- and low-grade gliomas, medulloblastomas, ependymomas, germ cell tumors, and meningiomas. Pathogenic or likely pathogenic variants were identified in 3/13 patients (23.1%), while 10/13 (76.9%) carried variants of uncertain significance. A positive family history of cancer was present in 69.2% of cases. In selected patients, tumor sequencing confirmed the presence of HR-related alterations, supporting a biological role of these variants in tumorigenesis. Conclusions This study provides the first systematic description of germline RAD family gene variants in pediatric CNS tumors. Although limited by cohort size and heterogeneity, our findings suggest that inherited defects in HR may contribute to genomic instability and cancer predisposition in a subset of pediatric CNS tumor patients. Larger, dedicated studies integrating functional assays and familial segregation analyses are warranted to define the clinical relevance of RAD alterations and their implications for genetic counseling and surveillance.

The establishment of neuronal polarity, whereby somatodendritic and axonal cellular compartments are defined, is a critical determinant for the development of neuronal networks and patterning during neurogenesis. The axon initial segment (AIS), a key structure in the establishment of this polarity, is formed through interactions between the microtubule and actin cytoskeleton, Ankyrin G, TRIM46 and multiple transmembrane and perimembranous proteins. Here we implicate a component of the septin cytoskeleton, Septin-2, in the maintenance and function of the AIS through the study of mutations found in five unrelated human individuals and one mother-daughter duo with a majority presenting with cognitive impairment. Septins form octameric rods that assemble into higher order filamentous scaffolds driven by Septin-2 homodimerization. Mutant Septin-2 is predicted to impart a dominant negative blockade on septin octamers forming these structures by precluding Septin-2 homodimerization. Expression of mutant Septin-2 constructs in neurons leads to the disappearance of canonical hallmarks of the AIS. This includes loss of Ankyrin G in the AIS, aberrant localization of MAP2 within the distal axon, axonal shortening and electrophysiological hypoexcitability. We further show that Septin-2 binds to a neuron-specific domain of Ankyrin G, an interaction that is largely ablated by these mutations. These data establish a role for Septin-2 in the maintenance and function of the AIS and implicate cytoskeletal structures composed of septin oligomers in the establishment of higher cognitive functions in humans.

Abstract Background Pathogenic germline variants in MUTYH, a key component of the base excision repair (BER) pathway, are classically associated with MUTYH-associated polyposis and colorectal cancer. Increasing evidence suggests that monoallelic MUTYH variants may confer low-penetrance susceptibility to extraintestinal malignancies, including central nervous system (CNS) tumors. We assessed the prevalence and spectrum of germline MUTYH variants in a large cohort of pediatric and adolescent/young adult (AYA) patients with brain tumors. Methods We retrospectively reviewed 378 consecutive pediatric and AYA patients with primary CNS tumors diagnosed between 2011 and 2025 at a single institution. All patients underwent clinical exome sequencing including MUTYH. Tumors were classified according to the WHO CNS 5th edition. Variant interpretation followed ACMG criteria, and inheritance was assessed by trio-based sequencing when available. Somatic MUTYH alterations were explored by tumor NGS in a subset of cases. Results Germline MUTYH variants were identified in 15/378 patients (4.0%). Pathogenic/likely pathogenic (P/LP) variants were detected in 11/15 (73%), while 4/15 (27%) carried variants of uncertain significance. One patient harbored biallelic P/LP variants (p.Glu452del and p.Tyr76*). Among monoallelic P/LP carriers (n = 10), recurrent founder variants predominated, including p.Tyr179Cys–equivalent substitutions (n = 4) and p.Gly396Asp–equivalent substitutions (n = 5), with an additional p.Arg242His variant (n = 1). A positive family history of cancer was reported in 60% of variant-positive cases. Six patients (40%) also carried additional germline alterations in other cancer-predisposition genes. Conclusions In this single-institution pediatric/AYA CNS tumor cohort, 4.0% of patients carried germline MUTYH variants, predominantly monoallelic P/LP founder substitutions. These findings support inclusion of MUTYH in multigene germline testing for pediatric CNS tumors and warrant further studies to clarify its role as a low-penetrance susceptibility factor and potential modifier in neuro-oncogenesis.

INTRODUCTION:Genomic instability is common in cancer, driven by different mechanisms and often linked to disease stage and progression. Optical genome mapping (OGM) enables the detection of genome-wide balanced and unbalanced structural rearrangements (SRs), providing an overview of genomic complexity. AIMS:To explore the utility of OGM in brain tumour characterisation, we conducted a pilot study on a well-characterised series of 43, mostly paediatric, cases encompassing different histotypes and enriched for gene fusion-positive neoplasms (30 cases). RESULTS:SRs were observed in 37/43 samples and defined three genomic patterns based on their number and distribution across the chromosomes: SR-chromothripsis (11 cases), high SR-complexity (11 cases) and low SR-complexity (21 cases). Genomic complexity was also assessed according to the number of copy number alterations (CNA) and, to this end, in SR-chromothripsis samples, only CNAs affecting chromosomes not involved in chromothripsis were considered: absence of CNAs was found in 15 cases, 1-9 CNAs in 19 cases, 10-49 CNAs in three cases and ≥ 50 CNAs in six cases. When examining the relationship between SR and CNA, a positive correlation emerged between CNA burden and the number of chromosomes harbouring SR (Spearman's r = 0.588, p = 0.0001), while breakpoint number was weakly associated; chromothripsis occurred exclusively within low CNA-complexity groups (p = 0.0836). Genome complexity patterns correlated with tumour types, with diffuse high-grade gliomas exhibiting high complexity, whereas infant-type hemispheric gliomas, most low-grade gliomas and embryonal tumours showed low complexity profiles. Importantly, SR-chromothripsis cases corresponded to low/intermediate-grade fusion-driven tumours, with balanced/nearly balanced CNA profiles. OGM allowed the detection of gene fusions in 24/30 cases, failing in six. When integrated with RNA sequencing, OGM unveiled the mechanisms underlying gene fusion formation in all cases: chromothripsis (11 cases), isolated chromosomal abnormalities (12 cases) and genome-wide alterations (seven cases). CONCLUSIONS:OGM reveals distinct genomic complexity patterns and refines the definition of chromothripsis, improving insights into tumourigenic mechanisms.

Heterozygous loss-of-function variants in SUFU are associated with Gorlin syndrome (MIM:620343) and tumor predisposition, while biallelic missense variants underlie recessive Joubert syndrome (JS; MIM:617757). Interestingly, emerging evidence suggests that SUFU haploinsufficiency also contributes to neurodevelopmental disorders (NDDs). We investigated 5 unrelated families encompassing 4 singletons and 1 pair of monozygotic twins carrying heterozygous SUFU loss-of-function variants through clinical, neuropsychological, and neuroimaging assessments and compared their features with 47 published cases fulfilling NDDs inclusion criteria. A consistent neurodevelopmental phenotype was observed, characterized by global developmental delay, hypotonia, congenital ocular motor apraxia, macrocephaly, and mild cerebellar abnormalities, including vermian hypoplasia and posterior fossa anomalies, variably falling within the JS spectrum. Craniofacial features were variably observed in our cohort and are reported as supportive clinical observations. Intellectual disability was present in similar to 35% of affected individuals, while autism spectrum disorder and behavioral difficulties occurred less frequently. Notably, neoplastic manifestations were absent among our cases, indicating that oncological and neurodevelopmental outcomes may segregate independently. SUFU haploinsufficiency may underlie a distinct dominantly inherited neurodevelopmental syndrome spanning from isolated ocular motor apraxia to a mild Joubert-like phenotype with macrocephaly and variable cognitive/behavioral outcomes. These findings further refine the clinical description of the SUFU-related neurodevelopmental phenotype beyond tumor predisposition and support its inclusion in diagnostic panels for developmental delay, macrocephaly, and JS-spectrum disorders. Multidisciplinary surveillance, balancing neurodevelopmental and oncological aspects, is recommended.

Giant congenital melanocytic nevi (GCMN) are benign mosaic disorders that may give rise to various tumor types through incompletely understood mechanisms. We characterized a series of two melanomas and four mesenchymal tumors (two embryonal rhabdomyosarcomas, one small round cell sarcoma, one low-grade mesenchymal tumor) arising in GCMN. Median onset age was 3.5 years and 3 months for melanoma and mesenchymal tumor patients, respectively. Both melanoma patients succumbed within 27 months from diagnosis, whereas no patients progressed in the mesenchymal group (median follow-up 46 months). NRAS Q61, BRAF V600E mutation, and a novel in frame BIN1::BRAF fusion were detected in four, one, and one cases, respectively, with a higher variant allele frequency in the tumors compared with the matched GCMN. Copy number alterations and/or copy-neutral loss of heterozygosity were exclusively found in the tumors in all cases. An inactivating ASXL1 variant and an in-frame KDM5B::LPGAT1 fusion were identified in one melanoma; paternal disomy of 11p15.5 in both embryonal rhabdomyosarcomas. Mesenchymal tumors and melanomas showed distinct transcriptional profiles enriched in muscle and synapse organization and epidermal differentiation genes, respectively.

De novo KCNA3 variants cause a Developmental and Epileptic Encephalopathy (DEE). We describe a 14-year-old boy presenting with DEE and carrying a heterozygous de novo KCNA3 (NM_002232.4) variant (c.1433T>A, p.Val478Glu) and an inherited KCNQ3 (NM_004519.3) variant (c.1720C>T, p.Pro574Ser). Human dermal fibroblasts (HDFs) were isolated from the patient and an age-matched control. KCNA3 and KCNQ3 transcript levels were quantified by qRT-PCR, showing in patient-HDFs a reduction of 77