Laura Licchetta

Objective: Capillary microsampling offers a minimally invasive alternative to venipuncture for therapeutic drug monitoring (TDM) of anti‐seizure medications (ASMs). We evaluated the feasibility and reliability of volumetric absorptive microsampling (VAMS) and quantitative dried blood spot (qDBS) devices for ambulatory self‐collection and at‐home use in persons with epilepsy (PwE). Methods: PwE attending the Epilepsy Centre of the IRCCS‐Istituto delle Scienze Neurologiche di Bologna (Italy) were enrolled between October 2023 and October 2024. Participants performed supervised self‐collection using VAMS and qDBS in ambulatory setting and at‐home self‐collection with VAMS devices. Sample quality, delivery success, and patient‐reported outcomes (ease, pain, and clarity of instructions) were recorded. Carbamazepine, lacosamide, lamotrigine, and levetiracetam were quantified using a validated ultra‐high performance liquid chromatography‐tandem mass spectrometry (UHPLC–MS/MS) method. Reliability was assessed by comparing at‐home VAMS with ambulatory VAMS and cross‐validating qDBS with VAMS and venous plasma samples using Bland–Altman analysis and linear regression. Results: A total of 105 PwE (66% female, mean age 41 years) performed at‐home and ambulatory self‐collection using VAMS and qDBS devices. Most at‐home VAMS samples (88%) were received by the laboratory within 7 days, and 86.8% met high‐quality criteria. For lacosamide, lamotrigine, and levetiracetam, we found strong correlations between at‐home and ambulatory VAMS (Pearson's r ranging from .88 to .98) and low mean bias (60 years impacted sampling quality, particularly for qDBS ( p = .04). Significance: VAMS‐based self‐collection is feasible and reliable for at‐home TDM, provided that high‐quality sampling is ensured. qDBS represents a reliable alternative for ambulatory monitoring. Future work should focus on optimizing sampling procedures for older adults and improving logistics for home‐based microsampling.

In a clinical setting, exome sequencing (ES) with copy number variant (CNV) analysis is currently the most effective approach for developmental and epileptic encephalopathies (DEE). However, trio‐based ES is often not feasible in adults, its costs remain prohibitive in certain health care settings, and computational tools for CNV calling still lack sufficient accuracy. Chromosomal microarray (CMA) is indicated as a first‐tier test for CNV detection in patients with DEE, dysmorphisms, and comorbidities. We investigate the role of CNVs in the etiology of DEE in an adult cohort, to define the most appropriate diagnostic approach in this population. A total of 219 patients (male/female: 104/115) with undiagnosed DEE underwent array‐based comparative genomic hybridization/single nucleotide polymorphism arrays as adults. Causative CNVs were identified in 18 patients (8.2%); 14 were deletions (mean size ≈ 2.96 Mb), and four were duplications/triplications (mean size ≈ 3.63 Mb). Thirteen were responsible for known deletion/microduplication syndromes, and four were deletions involving haploinsufficient genes associated with epilepsy/neurodevelopmental disorders. For one deletion, population evidence, reports with overlapping deletions, and clinical databases support a likely pathogenic role. The mean age at CMA diagnosis was 32.4 ± 13 years. An additional 46 patients (21%) then received a molecular diagnosis through alternative approaches. Despite a diagnostic delay of 24.2 ± 14.5 years, CMA enabled a genetic diagnosis in 8.2% of our adult DEE patients, especially in those with dysmorphisms. The diagnostic yield rises to 10.4% when excluding patients diagnosed using other methods. A total of 66.7% of solved cases had direct implications from the diagnosis, supporting the clinical utility of CNV analysis inclusion in the diagnostic workflow for adult patients with DEE.

Objective: Neurodevelopmental disorders (NDDs) and epilepsy are often associated. Increasing evidence highlights a pivotal role for pathogenic variants in genes encoding synaptic scaffolding proteins. Within this group, TANC2 has recently been implicated in intellectual developmental disorder with autistic features and language delay, with or without seizures; however, its full clinical spectrum and contribution to specific epileptic encephalopathies remain incompletely defined. Methods: We describe the electroclinical and developmental features of three patients carrying truncating TANC2 variants identified through trio‐exome sequencing within the European collaborative platform NETRE. Clinical, neuropsychological, and EEG data were collected and compared with prior reports. Results: Case #1 met Lennox–Gastaut syndrome (LGS) criteria, showing early drug resistance followed by partial cognitive recovery and sustained seizure control on felbamate monotherapy. Case #2 presented with ASD and multiple seizure types—including focal, atypical absence, and tonic—finally reaching prolonged remission and borderline intellectual functioning. Case #3 showed early‐onset, drug‐resistant polymorphic seizures with persistent bifrontal epileptiform discharges and severe developmental impairment, consistent with an LGS‐like phenotype, with seizure freedom achieved under a limited polytherapy regimen. Significance: Our findings expand the electroclinical spectrum of TANC2 ‐related disorders, supporting a continuum ranging from NDD‐associated epilepsy to DEE, including LGS in selected patients. Plain Language Summary: TANC2 is a gene involved in brain development and synaptic function. Changes in this gene have been linked to neurodevelopmental disorders, autism, intellectual disability, and epilepsy. We describe three individuals with previously unreported truncating TANC2 variants and different epilepsy phenotypes, including one patient fulfilling criteria for Lennox–Gastaut syndrome (LGS) and another with LGS‐like features. Although seizures were initially difficult to treat in some cases, seizure control was eventually achieved. These findings expand the known clinical spectrum of TANC2 ‐related disorders and suggest that selected patients may have a more favorable seizure course than expected.

Objective: Variants in GABRG2 , encoding the γ2 subunit of the γ‐aminobutyric acid type A receptor, are linked to epilepsy phenotypes of varying severity, with gain‐of‐function (GoF) variants associated with the more severe phenotypes than loss‐of‐function variants. Here, we provide a comprehensive analysis of the early clinical features, motor, language, and eating abilities, and structural consequences of the recurrent GABRG2 p.(Ala106Thr) GoF variant, aiming to refine genotype–phenotype correlations and deepen the understanding of GoF‐associated GABRG2 disorders. Methods: Individuals were recruited through international collaborations, literature review, and patient advocacy groups. Clinical data were collected via standardized interviews and physician reports. Functional effects were assessed using electrophysiological recordings. Structural modeling was performed using homology‐based approaches to evaluate conformational changes. Results: We collected 23 unrelated individuals harboring the GABRG2 p.(Ala106Thr) variant. Symptoms began with early dysphagia (median onset age = 1 month), hypotonia, seizures (median onset age = 2 months), and hyperkinetic movement disorders (median onset age = 3 months). Epilepsy was present in 91% of the individuals, 71% featuring a developmental and epileptic encephalopathy; 33% of the individuals with epilepsy achieved seizure freedom during the follow‐up. All individuals exhibited moderate to severe cognitive/neurodevelopmental impairment, with profound deficits in motor, language, and other domains. Functional analysis confirmed a GoF effect of the p.(Ala106Thr) variant. By contrast, another variant affecting the same residue, p.(Ala106Pro), which is observed in population databases, was functionally characterized as neutral. Structural modeling indicated that the p.(Ala106Thr) variant may enhance receptor gating by facilitating a hydrogen bond between the extracellular and transmembrane domains, a mechanism not observed with the p.(Ala106Pro) variant. Significance: Although some individuals exhibited severe developmental impairment without epilepsy, our findings demonstrate that the recurrent GABRG2 p.(Ala106Thr) GoF variant is consistently associated with a severe neurodevelopmental phenotype. Despite its profound clinical impact, in silico tools consistently predict this variant as benign, highlighting a critical gap in current predictive models.

Objective: To characterize the clinical, electroencephalographic, and genetic features of epilepsies featuring absence seizures within monogenic etiology, highlighting the diagnostic, treatment and prognostic implications. Methods: We conducted a retrospective, multicenter study including patients with monogenic epilepsies and electroencephalography (EEG)–documented absence seizures. We analyzed clinical data, electroclinical findings, neurodevelopmental outcomes, and treatment responses through standardized questionnaires and medical records. We classified genetic variants according to American College of Medical Genetics and Genomics (ACMG) guidelines and performed univariate and multivariate analyses to identify predictors of developmental outcomes. Results: We included 160 patients (111 female; median age at last follow‐up: 13 years) with absence seizures and confirmed pathogenic or likely pathogenic monogenic variants. The most frequently implicated genes were SLC2A1, SLC6A1, SYNGAP1 , CHD2, and SCN1A . Four genes— HESX1, NCKAP1, SON, STARD9 —had not been previously associated with absence seizures. In 35% of patients, absence seizures were the only seizure type and in 67% were the initial manifestation. Atypical features included irregular EEG discharges (56%) eyelid myoclonia (42%), and automatisms (33%). Early‐onset (before age 3) seizures occurred in 58% and was significantly associated with atypical features ( p < .03). Using existing International League Against Epilepsy (ILAE) epilepsy syndrome classification, 60% of patients could not be classified. Developmental delay occurred in 54%, intellectual disability in 65%, and other neurodevelopmental comorbidities in 49%. Predictors of poor developmental outcomes included early developmental delay, drug‐resistant epilepsy, and early absence onset. We found no difference in the prevalence of drug resistance across the various genetic etiologies. The most effective medications for absence seizures included valproate, ethosuximide, benzodiazepines, and lamotrigine. Disease‐specific therapies (e.g., ketogenic diet in SLC2A1 , stiripentol/fenfluramine in SCN1A) were effective in select cases. Significance: Absence seizures are a common manifestation of different monogenic epilepsies, often associated with early onset, atypical clinical and/or EEG features, developmental delay or drug resistance. Classification models should incorporate genetic data alongside electroclinical features, especially as next‐generation sequencing is increasingly used.

We report two cases of dual genetic diagnoses involving Lafora disease (LD) and co‐occurring neurodevelopmental disorders caused by pathogenic variants in TRIO and SHANK3 , respectively. LD is an ultra‐rare, autosomal recessive, severe form of progressive myoclonus epilepsy affecting previously healthy children or adolescents. In both patients, the presence of developmental delay, intellectual disability, and behavioral abnormalities was consistent with a primary genetic disorder— TRIO ‐related neurodevelopmental disorder in one, and Phelan‐McDermid syndrome in the other. However, the onset of epilepsy with atypical features, coupled with progressive neurological decline in one patient and a positive family history of LD in the other, prompted the additional diagnosis of LD. These cases illustrate how overlapping clinical presentations can obscure the presence of concomitant genetic conditions, potentially delaying diagnosis and appropriate management. Our findings underscore the importance of considering dual diagnoses and show that phenotypical variability in ultra‐rare disorders such as LD may be influenced by concurrent genetic conditions. Plain Language Summary: This report describes two patients who have both Lafora disease, an ultra‐rare, progressive type of epilepsy, and other rare genetic disorders that affect development and behavior. In one case, the patient showed a progressive and unusual neurological deterioration, while the other had atypical epileptic seizures and a family history of Lafora disease. These cases highlight how different genetic conditions can share similar symptoms, making it difficult to identify all the issues a patient may have. Understanding these overlaps is important for proper diagnosis and treatment.

Objective: DYNC1H1 variants are involved on a disease spectrum from neuromuscular disorders to neurodevelopmental disorders. DYNC1H1‐related epilepsy has been reported in small cohorts. We dissect the electroclinical features of 34 patients harboring de novo DYNC1H1 pathogenic variants, identify subphenotypes on the DYNC1H1‐related epilepsy spectrum, and compare the genotype–phenotype correlations observed in our cohort with the literature.Methods: Patients harboring de novo DYNC1H1 pathogenic variants were recruited through international collaborations. Clinical data were retrospectively collected. Latent class analysis was performed to identify subphenotypes. Multivariable binary logistic regression analysis was applied to investigate the association with DYNC1H1 protein domains.Results: DYNC1H1‐related epilepsy presented with infantile epileptic spasms syndrome (IESS) in 17 subjects (50%), and in 25% of these individuals the epileptic phenotype evolved into Lennox–Gastaut syndrome (LGS). In 12 patients (35%), focal onset epilepsy was defined. In two patients, the epileptic phenotype consisted of generalized myoclonic epilepsy, with a progressive phenotype in one individual harboring a frameshift variant. In approximately 60% of our cohort, seizures were drug‐resistant. Malformations of cortical development were noticed in 79% of our patients, mostly on the lissencephaly–pachygyria spectrum, particularly with posterior predominance in a half of them. Midline and infratentorial abnormalities were additionally reported in 45% and 27% of subjects. We have identified three main classes of subphenotypes on the DYNC1H1‐related epilepsy spectrum.Significance: We propose a classification in which pathogenic de novo DYNC1H1 variants feature drug‐resistant IESS in half of cases with potential evolution to LGS (Class 1), developmental and epileptic encephalopathy other than IESS and LGS (Class 2), or less severe focal or genetic generalized epilepsy including a progressive phenotype (Class 3). We observed an association between stalk domain variants and Class 1 phenotypes. The variants p.Arg309His and p.Arg1962His were common and associated with Class 1 subphenotype in our cohort. These findings may aid genetic counseling of patients with DYNC1H1‐related epilepsy.

Objective: YWHAG variant alleles have been associated with a rare disease trait whose clinical synopsis includes an early onset epileptic encephalopathy with predominantly myoclonic seizures, developmental delay/intellectual disability, and facial dysmorphisms. Through description of a large cohort, which doubles the number of reported patients, we further delineate the spectrum of YWHAG‐related epilepsy.Methods: We included in this study 24 patients, 21 new and three previously described, with pathogenic/likely pathogenic variants in YWHAG. We extended the analysis of clinical, electroencephalographic, brain magnetic resonance imaging, and molecular genetic information to 24 previously published patients.Results: The phenotypic spectrum of YWHAG‐related disorders ranges from mild developmental delay to developmental and epileptic encephalopathy (DEE). Epilepsy onset is in the first 2 years of life. Seizure freedom can be achieved in half of the patients (13/24, 54%). Intellectual disability (23/24, 96%), behavioral disorders (18/24, 75%), neurological signs (13/24, 54%), and dysmorphisms (6/24, 25%) are common. A genotype–phenotype correlation emerged, as DEE is more represented in patients with missense variants located in the ligand‐binding domain than in those with truncating or missense variants in other domains (90% vs. 19%, p < .001).Significance: This study suggests that pathogenic YWHAG variants cause a wide range of clinical presentations with variable severity, ranging from mild developmental delay to DEE. In this allelic series, a genotype–phenotype correlation begins to emerge, potentially providing prognostic information for clinical management and genetic counseling.

Background: Biallelic pathogenic variants in the mitochondrial prolyl‐tRNA synthetase 2 gene (PARS2, OMIM * 612036) have been associated with Developmental and Epileptic Encephalopathy‐75 (DEE‐75, MIM #618437). This condition is typically characterized by early‐onset refractory infantile spasms with hypsarrhythmia, intellectual disability, microcephaly, cerebral atrophy with hypomyelination, lactic acidemia, and cardiomyopathy. Most affected individuals do not survive beyond the age of 10 years.Methods: We describe a patient with early‐onset DEE, consistently showing an EEG pattern of Spike‐and‐Wave Activation in Sleep (SWAS) since childhood. The patient underwent extensive clinical, metabolic and genetic investigations, including whole exome sequencing (WES).Results: WES analysis identified compound heterozygous variants in PARS2 that have been already reported as pathogenic. A literature review of PARS2‐associated DEE, focusing mainly on the electroclinical phenotype, did not reveal the association of SWAS with pathogenic variants in PARS2.Notably, unlike previously reported cases with the same genotype, this patient had longer survival without cardiac involvement or lactic acidosis, suggesting potential genetic modifiers contributing to disease variability.Conclusion: These findings widen the genetic heterogeneity of DEE‐SWAS, including PARS2 as a causative gene in this syndromic entity, and highlight the importance of prolonged sleep EEG recording for the recognition of SWAS as a possible electroclinical evolution of PARS2‐related DEE.

Cryohydrocytosis is a form of stomatocytosis characterized by the leakage of sodium and potassium from red blood cells at low temperatures, characterized by pseudohyperkalemia. Stomatin‐deficient cryohydrocytosis is an extremely rare variant that only recently has been related to pathogenic variants in the SLC2A1 gene, encoding the main glucose transporter of the blood–brain barrier and red blood cells, GLUT1. It follows that GLUT1 deficiency syndrome, a rare but significant cause of metabolic epilepsy, may present with stomatin‐deficient cryohydrocytosis, although this correlation has only been reported in a few instances. We present the case of a patient carrying a novel de novo SLC2A1 pathogenic variant presenting with GLUT1 deficiency syndrome, pseudohyperkalemia, and splenomegaly consistent with cryohydrocytosis. We also review the previously reported cases of stomatin‐deficient cryohydrocytosis in the literature. As highlighted by our case, elevated potassium levels are a cause of concern, and GLUT1 deficiency syndrome patients are thus at risk of being subjected to unnecessary examinations; pseudohyperkalemia may be underrecognized in clinical practice.