Inferring disease course from differential exon usage in the wide titinopathy spectrum.

OBJECTIVE: Biallelic titin truncating variants (TTNtv) have been associated with a wide phenotypic spectrum, ranging from complex prenatal muscle diseases with dysmorphic features to adult-onset limb-girdle muscular dystrophy, with or without cardiac involvement. Given the size and complexity of TTN, reaching an unequivocal molecular diagnosis and precise disease prognosis remains challenging. METHODS: In this case series, 12 unpublished cases and one already published case with biallelic TTNtv were collected from multiple international medical centers between November 2022 and September 2023. TTN mutations were detected through exome or genome sequencing. Information about familial and personal clinical history was collected in a standardized form. RNA-sequencing and analysis of TTN exon usage were performed on an internal sample cohort including postnatal skeletal muscles, fetal skeletal muscles, postnatal heart muscles, and fetal heart muscles. In addition, publicly available RNA-sequencing data was retrieved from ENCODE. RESULTS: We generated new RNA-seq data on TTN exons and identified genotype-phenotype correlations with prognostic implications for each titinopathy patient (whether worsening or improving in prenatal and postnatal life) using percentage spliced in (PSI) data for the involved exons. Interestingly, thanks to exon usage, we were also able to rule out a titinopathy diagnosis in one prenatal case. INTERPRETATION: This study demonstrates that exon usage provides valuable insights for a more exhaustive clinical interpretation of TTNtv; additionally, it may serve as a model for implementing personalized medicine in many other genetic diseases, since most genes undergo alternative splicing.

Precision Medicine in Angelman Syndrome.

Angelman syndrome (AS) is a rare neurogenetic disorder caused by a loss of function of UBE3A on the maternal allele. Clinical features include severe neurodevelopmental delay, epilepsy, sleep disturbances, and behavioral disorders. Therapy currently evolves from conventional symptomatic, supportive, and antiseizure treatments toward alteration of mRNA expression, which is subject of several ongoing clinical trials.This article will provide an overview of clinical research and therapeutic approaches on AS.

Clinical features and genotype-phenotype correlations in epilepsy patients with de novo DYNC1H1 variants.

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.

Spectrum of NMDA Receptor Variants in Neurodevelopmental Disorders and Epilepsy.

N-methyl-D-aspartate receptors (NMDAR) are ligand-gated ion channels mediating excitatory neurotransmission and are important for normal brain development, cognitive abilities, and motor functions. Pathogenic variants in the Glutamate receptor Ionotropic N-methyl-D-aspartate (GRIN) genes (GRIN1, GRIN2A-D) encoding NMDAR subunits have been associated with a wide spectrum of neurodevelopmental disorders and epilepsies ranging from treatable focal epilepsies to devastating early-onset developmental and epileptic encephalopathies. Genetic variants in NMDA receptor genes can cause a range of complex alterations to receptor properties resulting in various degrees of loss-of-function, gain-of-function, or mixtures thereof. Understanding how genetic variants affect the function of the receptors, therefore, represents an important first step in the ongoing development towards targeted therapies. Currently, targeted treatment options for GRIN-related diseases are limited. However, treatment with memantine has been reported to significantly reduce seizure frequency in a few individuals with developmental and epileptic encephalopathies harboring de novo gain-of-function GRIN2A missense variants, and supplementary treatment with L-serine has been associated with improved motor and cognitive performance as well as reduced seizure frequency in patients with GRIN2B loss-of-function missense variants as well as GRIN2A and GRIN2B null variants.

Expanded phenotypic spectrum of neurodevelopmental and neurodegenerative disorder Bryant-Li-Bhoj syndrome with 38 additional individuals.

Bryant-Li-Bhoj syndrome (BLBS), which became OMIM-classified in 2022 (OMIM: 619720, 619721), is caused by germline variants in the two genes that encode histone H3.3 (H3-3A/H3F3A and H3-3B/H3F3B) [1-4]. This syndrome is characterized by developmental delay/intellectual disability, craniofacial anomalies, hyper/hypotonia, and abnormal neuroimaging [1, 5]. BLBS was initially categorized as a progressive neurodegenerative syndrome caused by de novo heterozygous variants in either H3-3A or H3-3B [1-4]. Here, we analyze the data of the 58 previously published individuals along 38 unpublished, unrelated individuals. In this larger cohort of 96 people, we identify causative missense, synonymous, and stop-loss variants. We also expand upon the phenotypic characterization by elaborating on the neurodevelopmental component of BLBS. Notably, phenotypic heterogeneity was present even amongst individuals harboring the same variant. To explore the complex phenotypic variation in this expanded cohort, the relationships between syndromic phenotypes with three variables of interest were interrogated: sex, gene containing the causative variant, and variant location in the H3.3 protein. While specific genotype-phenotype correlations have not been conclusively delineated, the results presented here suggest that the location of the variants within the H3.3 protein and the affected gene (H3-3A or H3-3B) contribute more to the severity of distinct phenotypes than sex. Since these variables do not account for all BLBS phenotypic variability, these findings suggest that additional factors may play a role in modifying the phenotypes of affected individuals. Histones are poised at the interface of genetics and epigenetics, highlighting the potential role for gene-environment interactions and the importance of future research.

Generation of two patient specific GABRD variants and their isogenic controls for modeling epilepsy.

Developmental and epileptic encephalopathies (DEEs) are early-onset conditions that cause intractable seizures and developmental delays. Missense variants in Gamma-aminobutyric acid type A receptor (GABAAR) subunits commonly cause DEEs. Ahring et al. (2022) showed a variant in the gene that encodes the delta subunit (GABRD) is strongly associated with the gain-of-function of extrasynaptic GABAAR. Here, we report the generation of two patient-specific human induced pluripotent stem cells (hiPSC) lines with (i) a de novo variant and (ii) a maternal variant, both for the pathogenic GABRD c.872 C>T, (p.T291I). The variants in the generated cell line were corrected using the CRISPR-Cas9 gene editing technique (respective isogenic control lines).

Gain-of-function and loss-of-function variants in GRIA3 lead to distinct neurodevelopmental phenotypes.

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs) mediate fast excitatory neurotransmission in the brain. AMPARs form by homo- or heteromeric assembly of subunits encoded by the GRIA1-GRIA4 genes, of which only GRIA3 is X-chromosomal. Increasing numbers of GRIA3 missense variants are reported in patients with neurodevelopmental disorders (NDD), but only a few have been examined functionally. Here, we evaluated the impact on AMPAR function of one frameshift and 43 rare missense GRIA3 variants identified in patients with NDD by electrophysiological assays. Thirty-one variants alter receptor function and show loss-of-function (LoF) or gain-of-function (GoF) properties, whereas 13 appeared neutral. We collected detailed clinical data from 25 patients (from 23 families) harbouring 17 of these variants. All patients had global developmental impairment, mostly moderate (9/25) or severe (12/25). Twelve patients had seizures, including focal motor (6/12), unknown onset motor (4/12), focal impaired awareness (1/12), (atypical) absence (2/12), myoclonic (5/12), and generalized tonic-clonic (1/12) or atonic (1/12) seizures. The epilepsy syndrome was classified as developmental and epileptic encephalopathy in eight patients, developmental encephalopathy without seizures in 13 patients, and intellectual disability with epilepsy in four patients. Limb muscular hypotonia was reported in 13/25, and hypertonia in 10/25. Movement disorders were reported in 14/25, with hyperekplexia or non-epileptic erratic myoclonus being the most prevalent feature (8/25). Correlating receptor functional phenotype with clinical features revealed clinical features for GRIA3-associated NDDs and distinct NDD phenotypes for LoF and GoF variants. GoF variants were associated with more severe outcomes: patients were younger at the time of seizure onset (median age one month), hypertonic, and more often had movement disorders, including hyperekplexia. Patients with LoF variants were older at the time of seizure onset (median age 16 months), hypotonic, and had sleeping disturbances. LoF and GoF variants were disease-causing in both sexes but affected males often carried de novo or hemizygous LoF variants inherited from healthy mothers, whereas all but one affected females had de novo heterozygous GoF variants.

Loss of Grin2a causes a transient delay in the electrophysiological maturation of hippocampal parvalbumin interneurons.

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ionotropic glutamate receptors that mediate a calcium-permeable component to fast excitatory neurotransmission. NMDARs are heterotetrameric assemblies of two obligate GluN1 subunits (GRIN1) and two GluN2 subunits (GRIN2A-GRIN2D). Sequencing data shows that 43% (297/679) of all currently known NMDAR disease-associated genetic variants are within the GRIN2A gene, which encodes the GluN2A subunit. Here, we show that unlike missense GRIN2A variants, individuals affected with disease-associated null GRIN2A variants demonstrate a transient period of seizure susceptibility that begins during infancy and diminishes near adolescence. We show increased circuit excitability and CA1 pyramidal cell output in juvenile mice of both Grin2a+/- and Grin2a-/- mice. These alterations in somatic spiking are not due to global upregulation of most Grin genes (including Grin2b). Deeper evaluation of the developing CA1 circuit led us to uncover age- and Grin2a gene dosing-dependent transient delays in the electrophysiological maturation programs of parvalbumin (PV) interneurons. We report that Grin2a+/+ mice reach PV cell electrophysiological maturation between the neonatal and juvenile neurodevelopmental timepoints, with Grin2a+/- mice not reaching PV cell electrophysiological maturation until preadolescence, and Grin2a-/- mice not reaching PV cell electrophysiological maturation until adulthood. Overall, these data may represent a molecular mechanism describing the transient nature of seizure susceptibility in disease-associated null GRIN2A patients.

CUX1-related neurodevelopmental disorder: deep insights into phenotype-genotype spectrum and underlying pathology.

Heterozygous, pathogenic CUX1 variants are associated with global developmental delay or intellectual disability. This study delineates the clinical presentation in an extended cohort and investigates the molecular mechanism underlying the disorder in a Cux1+/- mouse model. Through international collaboration, we assembled the phenotypic and molecular information for 34 individuals (23 unpublished individuals). We analyze brain CUX1 expression and susceptibility to epilepsy in Cux1+/- mice. We describe 34 individuals, from which 30 were unrelated, with 26 different null and four missense variants. The leading symptoms were mild to moderate delayed speech and motor development and borderline to moderate intellectual disability. Additional symptoms were muscular hypotonia, seizures, joint laxity, and abnormalities of the forehead. In Cux1+/- mice, we found delayed growth, histologically normal brains, and increased susceptibility to seizures. In Cux1+/- brains, the expression of Cux1 transcripts was half of WT animals. Expression of CUX1 proteins was reduced, although in early postnatal animals significantly more than in adults. In summary, disease-causing CUX1 variants result in a non-syndromic phenotype of developmental delay and intellectual disability. In some individuals, this phenotype ameliorates with age, resulting in a clinical catch-up and normal IQ in adulthood. The post-transcriptional balance of CUX1 expression in the heterozygous brain at late developmental stages appears important for this favorable clinical course.

Expanding SPTAN1 monoallelic variant associated disorders: From epileptic encephalopathy to pure spastic paraplegia and ataxia.

PURPOSE: Nonerythrocytic αII-spectrin (SPTAN1) variants have been previously associated with intellectual disability and epilepsy. We conducted this study to delineate the phenotypic spectrum of SPTAN1 variants. METHODS: We carried out SPTAN1 gene enrichment analysis in the rare disease component of the 100,000 Genomes Project and screened 100,000 Genomes Project, DECIPHER database, and GeneMatcher to identify individuals with SPTAN1 variants. Functional studies were performed on fibroblasts from 2 patients. RESULTS: Statistically significant enrichment of rare (minor allele frequency < 1 × 10-5) probably damaging SPTAN1 variants was identified in families with hereditary ataxia (HA) or hereditary spastic paraplegia (HSP) (12/1142 cases vs 52/23,847 controls, p = 2.8 × 10-5). We identified 31 individuals carrying SPTAN1 heterozygous variants or deletions. A total of 10 patients presented with pure or complex HSP/HA. The remaining 21 patients had developmental delay and seizures. Irregular αII-spectrin aggregation was noted in fibroblasts derived from 2 patients with p.(Arg19Trp) and p.(Glu2207del) variants. CONCLUSION: We found that SPTAN1 is a genetic cause of neurodevelopmental disorder, which we classified into 3 distinct subgroups. The first comprises developmental epileptic encephalopathy. The second group exhibits milder phenotypes of developmental delay with or without seizures. The final group accounts for patients with pure or complex HSP/HA.

Current practice in diagnostic genetic testing of the epilepsies

Epilepsy genetics is a rapidly developing field, in which novel disease-associated genes, novel mechanisms associated with epilepsy, and precision medicine approaches are continuously being identified. In the past decade, advances in genomic knowledge and analysis platforms have begun to make clinical genetic testing accessible for, in principle, people of all ages with epilepsy. For this reason, the Genetics Commission of the International League Against Epilepsy (ILAE) presents this update on clinical genetic testing practice, including current techniques, indications, yield of genetic testing, recommendations for pre- and post-test counseling, and follow-up after genetic testing is completed. We acknowledge that the resources vary across different settings but highlight that genetic diagnostic testing for epilepsy should be prioritized when the likelihood of an informative finding is high. Results of genetic testing, in particular the identification of causative genetic variants, are likely to improve individual care. We emphasize the importance of genetic testing for individuals with epilepsy as we enter the era of precision therapy.

L-Serine Treatment is Associated with Improvements in Behavior, EEG, and Seizure Frequency in Individuals with GRIN-Related Disorders Due to Null Variants.

Pathogenic missense variants in GRIN2A and GRIN2B may result in gain or loss of function (GoF/LoF) of the N-methyl-D-aspartate receptor (NMDAR). This observation gave rise to the hypothesis of successfully treating GRIN-related disorders due to LoF variants with co-agonists of the NMDAR. In this respect, we describe a retrospectively collected series of ten individuals with GRIN2A- or GRIN2B-related disorders who were treated with L-serine, each within an independent n-of-1 trial. Our cohort comprises one individual with a LoF missense variant with clinical improvements confirming the above hypothesis and replicating a previous n-of-1 trial. A second individual with a GoF missense variant was erroneously treated with L-serine and experienced immediate temporary behavioral deterioration further supporting the supposed functional pathomechanism. Eight additional individuals with null variants (that had been interpreted as loss-of-function variants despite not being missense) again showed clinical improvements. Among all nine individuals with LoF missense or null variants, L-serine treatment was associated with improvements in behavior in eight (89%), in development in four (44%), and/or in EEG or seizure frequency in four (44%). None of these nine individuals experienced side effects or adverse findings in the context of L-serine treatment. In summary, we describe the first evidence that L-serine treatment may not only be associated with clinical improvements in GRIN-related disorders due to LoF missense but particularly also null variants.

Gain-of-function variants in GABRD reveal a novel pathway for neurodevelopmental disorders and epilepsy.

A potential link between GABRD encoding the δ subunit of extrasynaptic GABAA receptors and neurodevelopmental disorders has largely been disregarded due to conflicting conclusions from early studies. However, we identified seven heterozygous missense GABRD variants in 10 patients with neurodevelopmental disorders and generalized epilepsy. One variant occurred in two sibs of healthy parents with presumed somatic mosaicism, another segregated with the disease in three affected family members, and the remaining five occurred de novo in sporadic patients. Electrophysiological measurements were used to determine the functional consequence of the seven missense δ subunit variants in receptor combinations of α1ß3δ and α4ß2δ GABAA receptors. This was accompanied by analysis of electroclinical phenotypes of the affected individuals. We determined that five of the seven variants caused altered function of the resulting α1ß3δ and α4ß2δ GABAA receptors. Surprisingly, four of the five variants led to gain-of-function effects, whereas one led to a loss-of-function effect. The stark differences between the gain-of-function and loss-of function effects were mirrored by the clinical phenotypes. Six patients with gain-of-function variants shared common phenotypes: neurodevelopmental disorders with behavioural issues, various degrees of intellectual disability, generalized epilepsy with atypical absences and generalized myoclonic and/or bilateral tonic-clonic seizures. The EEG showed qualitative analogies among the different gain-of-function variant carriers consisting of focal slowing in the occipital regions often preceding irregular generalized epileptiform discharges, with frontal predominance. In contrast, the one patient carrying a loss-of-function variant had normal intelligence and no seizure history, but has a diagnosis of autism spectrum disorder and suffers from elevated internalizing psychiatric symptoms. We hypothesize that increase in tonic GABA-evoked current levels mediated by δ-containing extrasynaptic GABAA receptors lead to abnormal neurotransmission, which represent a novel mechanism for severe neurodevelopmental disorders. In support of this, the electroclinical findings for the gain-of-function GABRD variants resemble the phenotypic spectrum reported in patients with missense SLC6A1 (GABA uptake transporter) variants. This also indicates that the phenomenon of extrasynaptic receptor overactivity is observed in a broader range of patients with neurodevelopmental disorders, because SLC6A1 loss-of-function variants also lead to overactive extrasynaptic δ-containing GABAA receptors. These findings have implications when selecting potential treatment options, as a substantial portion of available antiseizure medication act by enhancing GABAergic function either directly or indirectly, which could exacerbate symptoms in patients with gain-of-function GABRD variants.

Prenatal phenotype of PNKP-related primary microcephaly associated with variants affecting both the FHA and phosphatase domain.

Biallelic PNKP variants cause heterogeneous disorders ranging from neurodevelopmental disorder with microcephaly/seizures to adult-onset Charcot-Marie-Tooth disease. To date, only postnatal descriptions exist. We present the first prenatal diagnosis of PNKP-related primary microcephaly. Pathological examination of a male fetus in the 18th gestational week revealed micrencephaly with extracerebral malformations and thus presumed syndromic microcephaly. A recessive disorder was suspected because of previous pregnancy termination for similar abnormalities. Prenatal trio-exome sequencing identified compound heterozygosity for the PNKP variants c.498G>A, p.[(=),0?] and c.302C>T, p.(Pro101Leu). Segregation confirmed both variants in the sister fetus. Through RNA analyses, we characterized exon 4 skipping affecting the PNKP forkhead-associated (FHA) and phosphatase domains (p.Leu67_Lys166del) as the predominant effect of the paternal c.498G>A variant. We retrospectively investigated two unrelated individuals diagnosed with biallelic PNKP-variants to compare prenatal/postnatal phenotypes. Both carry the splice donor variant c.1029+2T>C in trans with a variant in the FHA domain (c.311T>C, p.(Leu104Pro); c.151G>C, p.(Val51Leu)). RNA-seq showed complex splicing for c.1029+2T>C and c.151G>C. Structural modeling revealed significant clustering of missense variants in the FHA domain with variants generating structural damage. Our clinical description extends the PNKP-continuum to the prenatal stage. Investigating possible PNKP-variant effects using RNA and structural modeling, we highlight the mutational complexity and exemplify a PNKP-variant characterization framework.

Genotype-phenotype correlations in SCN8A-related disorders reveal prognostic and therapeutic implications.

We report detailed functional analyses and genotype-phenotype correlations in 392 individuals carrying disease-causing variants in SCN8A, encoding the voltage-gated Na+ channel Nav1.6, with the aim of describing clinical phenotypes related to functional effects. Six different clinical subgroups were identified: Group 1, benign familial infantile epilepsy (n = 15, normal cognition, treatable seizures); Group 2, intermediate epilepsy (n = 33, mild intellectual disability, partially pharmaco-responsive); Group 3, developmental and epileptic encephalopathy (n = 177, severe intellectual disability, majority pharmaco-resistant); Group 4, generalized epilepsy (n = 20, mild to moderate intellectual disability, frequently with absence seizures); Group 5, unclassifiable epilepsy (n = 127); and Group 6, neurodevelopmental disorder without epilepsy (n = 20, mild to moderate intellectual disability). Those in Groups 1-3 presented with focal or multifocal seizures (median age of onset: 4 months) and focal epileptiform discharges, whereas the onset of seizures in patients with generalized epilepsy was later (median: 42 months) with generalized epileptiform discharges. We performed functional studies expressing missense variants in ND7/23 neuroblastoma cells and primary neuronal cultures using recombinant tetrodotoxin-insensitive human Nav1.6 channels and whole-cell patch-clamping. Two variants causing developmental and epileptic encephalopathy showed a strong gain-of-function (hyperpolarizing shift of steady-state activation, strongly increased neuronal firing rate) and one variant causing benign familial infantile epilepsy or intermediate epilepsy showed a mild gain-of-function (defective fast inactivation, less increased firing). In contrast, all three variants causing generalized epilepsy induced a loss-of-function (reduced current amplitudes, depolarizing shift of steady-state activation, reduced neuronal firing). Functional effects were known for 170 individuals. All 136 individuals carrying a functionally tested gain-of-function variant had either focal (n = 97, Groups 1-3) or unclassifiable (n = 39) epilepsy, whereas 34 individuals with a loss-of-function variant had either generalized (n = 14), no (n = 11) or unclassifiable (n = 6) epilepsy; only three had developmental and epileptic encephalopathy. Computational modelling in the gain-of-function group revealed a significant correlation between the severity of the electrophysiological and clinical phenotypes. Gain-of-function variant carriers responded significantly better to sodium channel blockers than to other anti-seizure medications, and the same applied for all individuals in Groups 1-3. In conclusion, our data reveal clear genotype-phenotype correlations between age at seizure onset, type of epilepsy and gain- or loss-of-function effects of SCN8A variants. Generalized epilepsy with absence seizures is the main epilepsy phenotype of loss-of-function variant carriers and the extent of the electrophysiological dysfunction of the gain-of-function variants is a main determinant of the severity of the clinical phenotype in focal epilepsies. Our pharmacological data indicate that sodium channel blockers present a treatment option in SCN8A-related focal epilepsy with onset in the first year of life.

Genetic testing in adults with developmental and epileptic encephalopathy - what do we know?

Knowledge of underlying genetic causes of developmental and epileptic encephalopathies (DEE) in adults is still limited when compared to the routine diagnostic approach in similarly affected children. A well-documented longitudinal study of adults with DEE is of utmost importance to understand the natural history of the respective entity. This information is of great value especially for genetic counselling of newly diagnosed children with identical genetic diagnoses and may impact treatment and management of affected individuals. In our meta-analysis we provide an overview of the most recurrent genetic findings across an adult DEE cohort (n=1,020). The gene mostly associated with a pathogenic or likely pathogenic variant in adult DEE is SCN1A, followed by MECP2 and CHD2. Studies employing exome sequencing and calling of both single nucleotide variants and copy number variants are associated with diagnostic yields of almost 50 %. Finally, we highlight three remarkable cases, each representing the oldest individual ever published with their genetic diagnosis, i. e., Angelman syndrome, Miller-Dieker syndrome, and CAMK2A-related disorder, and describe lessons learned from each of these adults.