Individualised human phenotype ontology gene panels improve clinical whole exome and genome sequencing analytical efficacy in a cohort of developmental and epileptic encephalopathies.

BACKGROUND: The majority of genetic epilepsies remain unsolved in terms of specific genotype. Phenotype-based genomic analyses have shown potential to strengthen genomic analysis in various ways, including improving analytical efficacy. METHODS: We have tested a standardised phenotyping method termed 'Phenomodels' for integrating deep-phenotyping information with our in-house developed clinical whole exome/genome sequencing analytical pipeline. Phenomodels includes a user-friendly epilepsy phenotyping template and an objective measure for selecting which template terms to include in individualised Human Phenotype Ontology (HPO) gene panels. In a pilot study of 38 previously solved cases of developmental and epileptic encephalopathies, we compared the sensitivity and specificity of the individualised HPO gene panels with the clinical epilepsy gene panel. RESULTS: The Phenomodels template showed high sensitivity for capturing relevant phenotypic information, where 37/38 individuals' HPO gene panels included the causative gene. The HPO gene panels also had far fewer variants to assess than the epilepsy gene panel. CONCLUSION: We have demonstrated a viable approach for incorporating standardised phenotype information into clinical genomic analyses, which may enable more efficient analysis.

[Epilepsy surgery seems underutilized in Sweden]. / Epilepsikirurgin är underutny­­ttjad ­ fler bör hänvisas till utredning.

Epilepsy surgery should be considered for individuals with drug-resistant focal epilepsy. The pre-surgical evaluation is highly multi-disciplinary and performed by a team consisting of neurologists, neurophysiologists, neurosurgeons, neuroradiologists, neuropsychologists, biomedical scientists, speech-language pathologists and nursing staff. The evaluation comprises of a meticulous medical history with focus on seizure semiology, a 3 Tesla MRI, ictal video-EEG, neuropsychological evaluation and sometimes also MEG, nTMS, fMRI, PET or SISCOM/ictal SPECT. Occasionally, invasive monitoring with intracranial electrodes is necessary. Surgical options in treatment of epilepsy range from open resections of epileptogenic areas to focal ablations and neurostimulation. There is evidence of epilepsy surgery being an effective treatment in carefully selected cases. Epilepsy surgery seems underutilized in Sweden and referrals for epilepsy surgery work-up need to increase.

Case report: Fatal outcome of pyridoxine-dependent epilepsy presenting as respiratory distress followed by a circulatory collapse.

Pyridoxine-dependent epilepsy is a rare autosomal recessive disease usually associated with neonatal seizures that do not respond to common antiseizure medications but are controlled by pyridoxine administration. Because the symptoms can mimic common neonatal disorders, the diagnosis can be initially missed or delayed. We report a fatal case of a boy who was initially diagnosed with respiratory distress, birth asphyxia, and persistent pulmonary hypertension and whose condition rapidly deteriorated during the first day of life.

Outcome at age 7 of epilepsy presenting in the first 2 years of life. A population-based study.

OBJECTIVE: Existing data suggest that epilepsy presenting in the first few years of life carries a worse prognosis than later onset. However, studies are few and methods differ, making interpretations of data uncertain. This study analyzes outcome at age 7 and potential prognostic factors in a well-characterized population-based cohort with epilepsy onset during the first 2 years of life. METHODS: An incidence cohort of 116 prospectively identified cases of epilepsy with seizure onset before age 2 years was described in Stödberg et al. (2020). Cases were originally retrieved from the Stockholm Incidence Registry of Epilepsy (SIRE), which registered all cases with a first unprovoked epileptic seizure from September 1, 2001, in Northern Stockholm. Data on treatment and outcome at age 7 years were collected from electronic medical records and through interviews with parents. Outcome and potential prognostic factors were analyzed with descriptive statistics and multivariable log binomial regression analysis. RESULTS: Eleven children (9.5%) died before age 7. Polytherapy was common. Epilepsy surgery was performed in two children. At age 7 years, 61 of 116 children (53%) had been seizure-free for the last 2 years or longer. Intellectual disability was diagnosed in 57 of 116 children (49%), autism spectrum disorder in 13 (11%), and cerebral palsy in 28 (24%). West syndrome had a similar seizure remission rate but a worse cognitive outcome. There was no difference in outcome between first and second year onset. Six predictors, including etiology, remained associated with two or more outcome variables after regression analysis. SIGNIFICANCE: About half of children with infantile-onset epilepsy will become seizure-free and half of them will have intellectual disability. Etiology was confirmed as a major independent predictor of outcome. Our study contributes to a more firm knowledge base when counseling parents of infants diagnosed with epilepsy.

Integration of whole genome sequencing into a healthcare setting: high diagnostic rates across multiple clinical entities in 3219 rare disease patients.

BACKGROUND: We report the findings from 4437 individuals (3219 patients and 1218 relatives) who have been analyzed by whole genome sequencing (WGS) at the Genomic Medicine Center Karolinska-Rare Diseases (GMCK-RD) since mid-2015. GMCK-RD represents a long-term collaborative initiative between Karolinska University Hospital and Science for Life Laboratory to establish advanced, genomics-based diagnostics in the Stockholm healthcare setting. METHODS: Our analysis covers detection and interpretation of SNVs, INDELs, uniparental disomy, CNVs, balanced structural variants, and short tandem repeat expansions. Visualization of results for clinical interpretation is carried out in Scout-a custom-developed decision support system. Results from both singleton (84%) and trio/family (16%) analyses are reported. Variant interpretation is done by 15 expert teams at the hospital involving staff from three clinics. For patients with complex phenotypes, data is shared between the teams. RESULTS: Overall, 40% of the patients received a molecular diagnosis ranging from 19 to 54% for specific disease groups. There was heterogeneity regarding causative genes (n = 754) with some of the most common ones being COL2A1 (n = 12; skeletal dysplasia), SCN1A (n = 8; epilepsy), and TNFRSF13B (n = 4; inborn errors of immunity). Some causative variants were recurrent, including previously known founder mutations, some novel mutations, and recurrent de novo mutations. Overall, GMCK-RD has resulted in a large number of patients receiving specific molecular diagnoses. Furthermore, negative cases have been included in research studies that have resulted in the discovery of 17 published, novel disease-causing genes. To facilitate the discovery of new disease genes, GMCK-RD has joined international data sharing initiatives, including ClinVar, UDNI, Beacon, and MatchMaker Exchange. CONCLUSIONS: Clinical WGS at GMCK-RD has provided molecular diagnoses to over 1200 individuals with a broad range of rare diseases. Consolidation and spread of this clinical-academic partnership will enable large-scale national collaboration.

De novo variants in SNAP25 cause an early-onset developmental and epileptic encephalopathy.

PURPOSE: This study aims to provide a comprehensive description of the phenotypic and genotypic spectrum of SNAP25 developmental and epileptic encephalopathy (SNAP25-DEE) by reviewing newly identified and previously reported individuals. METHODS: Individuals harboring heterozygous missense or loss-of-function variants in SNAP25 were assembled through collaboration with international colleagues, matchmaking platforms, and literature review. For each individual, detailed phenotyping, classification, and structural modeling of the identified variant were performed. RESULTS: The cohort comprises 23 individuals with pathogenic or likely pathogenic de novo variants in SNAP25. Intellectual disability and early-onset epilepsy were identified as the core symptoms of SNAP25-DEE, with recurrent findings of movement disorders, cerebral visual impairment, and brain atrophy. Structural modeling for all variants predicted possible functional defects concerning SNAP25 or impaired interaction with other components of the SNARE complex. CONCLUSION: We provide a comprehensive description of SNAP25-DEE with intellectual disability and early-onset epilepsy mostly occurring before the age of two years. These core symptoms and additional recurrent phenotypes show an overlap to genes encoding other components or associated proteins of the SNARE complex such as STX1B, STXBP1, or VAMP2. Thus, these findings advance the concept of a group of neurodevelopmental disorders that may be termed "SNAREopathies."

Epilepsy syndromes, etiologies, and the use of next-generation sequencing in epilepsy presenting in the first 2 years of life: A population-based study.

OBJECTIVE: Population-based data on epilepsy syndromes and etiologies in early onset epilepsy are scarce. The use of next-generation sequencing (NGS) has hitherto not been reported in this context. The aim of this study is to describe children with epilepsy onset before 2 years of age, and to explore to what degree whole exome and whole genome sequencing (WES/WGS) can help reveal a molecular genetic diagnosis. METHODS: Children presenting with a first unprovoked epileptic seizure before age 2 years and registered in the Stockholm Incidence Registry of Epilepsy (SIRE) between September 1, 2001 and December 31, 2006, were retrieved and their medical records up to age 7 years reviewed. Children who met the epilepsy criteria were included in the study cohort. WES/WGS was offered in cases of suspected genetic etiology regardless of whether a structural or metabolic diagnosis had been established. RESULTS: One hundred sixteen children were included, of which 88 had seizure onset during the first year of life and 28 during the second, corresponding to incidences of 139 and 42/100 000 person-years, respectively. An epilepsy syndrome could be diagnosed in 54% of cases, corresponding to a birth prevalence of 1/1100. Structural etiology was revealed in 34% of cases, a genetic cause in 20%, and altogether etiology was known in 65% of children. The highest diagnostic yield was seen in magnetic resonance imaging (MRI) with 65% revealing an etiology. WES/WGS was performed in 26/116 cases (22%), with a diagnostic yield of 58%. SIGNIFICANCE: Epilepsy syndromes can be diagnosed and etiologies revealed in a majority of early onset cases. NGS can identify a molecular diagnosis in a substantial number of children, and should be included in the work-up, especially in cases of epileptic encephalopathy, cerebral malformation, or metabolic disease without molecular diagnosis. A genetic diagnosis is essential to genetic counselling, prenatal diagnostics, and precision therapy.

SLC12A2 mutations cause NKCC1 deficiency with encephalopathy and impaired secretory epithelia.

OBJECTIVE: To describe the phenotype in 2 sisters with a rare constellation of neurologic symptoms and secretory impairments and to identify the etiology by the use of whole-genome sequencing (WGS). METHODS: After an extensive workup failed to reveal the cause of disease, in a girl with a previously not reported phenotype, WGS of the proband, her diseased older sister, an older healthy brother, and their parents was performed, and potentially pathogenic variants were analyzed. RESULTS: The proband and her older sister both presented with neonatal Staphylococcus aureus parotitis, apneas, disappearance of the Moro reflex, and hypotonia. The proband survived. Her brain MRI showed white matter and basal ganglia abnormalities, and CSF damage biomarkers were increased. At age 8 years, she exhibits a constellation of symptoms including severe neurodevelopmental disorder, hearing impairment, gastrointestinal problems, and a striking lack of tear fluid, saliva, and sweat. Her respiratory mucosa is dry with potentially life-threatening mucus plugging. Through WGS, 2 loss-of-function variants in SLC12A2 were identified that follow an autosomal recessive inheritance pattern. CONCLUSIONS: Taken together with a single previously reported case and the close resemblance to the phenotypes of corresponding mouse models, our study firmly establishes biallelic variants in SLC12A2 as causing human disease and adds data regarding the neurologic phenotype.

Diagnostic pitfalls in vitamin B6-dependent epilepsy caused by mutations in the PLPBP gene.

Vitamin B6-responsive epilepsies are a group of genetic disorders including ALDH7A1 deficiency, PNPO deficiency, and others, usually causing neonatal onset seizures resistant to treatment with common antiepileptic drugs. Recently, biallelic mutations in PLPBP were shown to be a novel cause of vitamin B6-dependent epilepsy with a variable phenotype. The different vitamin B6-responsive epilepsies can be detected and distinguished by their respective biomarkers and genetic analysis. Unfortunately, metabolic biomarkers for early detection and prognosis of PLPBP deficiency are currently still lacking. Here, we present data from two further patients with vitamin B6-dependent seizures caused by variants in PLPBP, including a novel missense variant, and compare their genotype and phenotypic presentation to previously described cases. Hyperglycinemia and hyperlactatemia are the most consistently observed biochemical abnormalities in pyridoxal phosphate homeostasis protein (PLPHP) deficient patients and were present in both patients in this report within the first days of life. Lactic acidemia, the neuroradiological, and clinical presentation led to misdiagnosis of a mitochondrial encephalopathy in two previously published cases with an early fatal course. Similarly, on the background of glycine elevation in plasma, glycine encephalopathy was wrongly adopted as diagnosis for a patient in our report. In this regard, lactic acidemia as well as hyperglycinemia appear to be diagnostic pitfalls in patients with vitamin B6-responsive epilepsies, including PLPHP deficiency. SYNOPSIS: In vitamin B6-responsive epilepsies, including PLPHP deficiency, there are several diagnostic pitfalls, including lactic acidemia as well as hyperglycinemia, highlighting the importance of a pyridoxine trial, and genetic testing.

Benign paroxysmal torticollis of infancy does not lead to neurological sequelae.

AIM: To elucidate the natural course of benign paroxysmal torticollis, the relationship of this disorder to migraine and other paroxysmal diseases, and to analyse candidate genes. METHOD: This was a case series of children with benign paroxysmal torticollis of infancy (BPTI) diagnosed from 1998 to 2005, at Astrid Lindgren Children's Hospital, Stockholm, Sweden. A neurological examination and a formalized motor assessment were performed from 2005 to 2007. At a second follow-up, in 2014 to 2015, the children and their parents were interviewed and candidate genes analysed. RESULTS: The mean age of the eight females and three males included in the second follow-up was 13 years 9 months (SD 2y 2mo). All motor assessments were normal. Five had developed migraine, abdominal migraine, and/or cyclic vomiting. Prophylactic treatment or migraine-specific medication during attacks were not needed. No paroxysmal tonic upgaze, benign paroxysmal vertigo, epilepsy, episodic ataxia, or paroxysmal dyskinesia was reported. Rare genetic variants in CACNA1A and ATP1A2 were found in two children. Five had a family history of migraine. INTERPRETATION: BPTI is transient and does not lead to neurological sequelae. Most children afflicted experience either a mild migraine or no paroxysmal disorder at all in their adolescence. Genetic variants in candidate genes were few, indicating potential genetic heterogeneity. WHAT THIS PAPER ADDS: After resolution of their benign paroxysmal torticollis of infancy (BPTI), children display no gross motor delay. Most adolescents who previously had BPTI have not developed migraine. No mutations in candidate genes, known to cause hemiplegic migraine, were found. Associated symptoms are often lacking during episodes of torticollis.

[Classification of seizures and epilepsies]. / Nya reviderade klassifikationer av epilepsianfall och sjukdomstyp - Den internationella epilepsiorganisationen har även lanserat en ny klinisk definition av epilepsi.

In 2017 the International League Against Epilepsy (ILAE) published position papers outlining new classifications of seizures and epilepsies. The aims of the new documents are to encompass advances in the field, provide a conceptual framework for future developments, and facilitate communication. In practice, the terminology is somewhat revised. For seizures the terms "partial" and "simple/complex" are replaced by "focal" and "aware/impaired awareness". The classification of a patient's epilepsy is based on seizures types (e.g. focal or generalized) as well as aetiology (e.g. structural, immune-mediated, genetic) and epilepsy syndrome. The present article describes the Swedish translation, endorsed by the Swedish Epilepsy Society.

[Diagnosing epileptic seizures and epilepsy]. / Utredning av epileptiska anfall och misstänkt epilepsi - Anamnes och vittnesbeskrivning är avgörande för rätt diagnos.

There are many episodic conditions which may be confused with epileptic seizures. The diagnosis of epileptic seizures is still dependent on a good history and witness report, as well as good knowledge of seizure semiology, and of the symptoms of a variety of differential diagnostic conditions. The principal differential diagnoses in adults and children are outlined in this review. The diagnostic problems do not exist only initially, so it is important to reconsider the diagnosis when deemed relevant. The possibility of home video recordings of recurring attacks may be helpful. Neuroimaging and interictal EEG cannot confirm or exclude epilepsy but can demonstrate relevant pathology and are of prognostic importance.

An LC-MS/MS-Based Method for the Quantification of Pyridox(am)ine 5'-Phosphate Oxidase Activity in Dried Blood Spots from Patients with Epilepsy.

We report the development of a rapid, simple, and robust LC-MS/MS-based enzyme assay using dried blood spots (DBS) for the diagnosis of pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency (OMIM 610090). PNPO deficiency leads to potentially fatal early infantile epileptic encephalopathy, severe developmental delay, and other features of neurological dysfunction. However, upon prompt treatment with high doses of vitamin B6, affected patients can have a normal developmental outcome. Prognosis of these patients is therefore reliant upon a rapid diagnosis. PNPO activity was quantified by measuring pyridoxal 5'-phosphate (PLP) concentrations in a DBS before and after a 30 min incubation with pyridoxine 5'-phosphate (PNP). Samples from 18 PNPO deficient patients (1 day-25 years), 13 children with other seizure disorders receiving B6 supplementation (1 month-16 years), and 37 child hospital controls (5 days-15 years) were analyzed. DBS from the PNPO-deficient samples showed enzyme activity levels lower than all samples from these two other groups as well as seven adult controls; no false positives or negatives were identified. The method was fully validated and is suitable for translation into the clinical diagnostic arena.

Genetic, Phenotypic, and Interferon Biomarker Status in ADAR1-Related Neurological Disease.

We investigated the genetic, phenotypic, and interferon status of 46 patients from 37 families with neurological disease due to mutations in ADAR1. The clinicoradiological phenotype encompassed a spectrum of Aicardi-Goutières syndrome, isolated bilateral striatal necrosis, spastic paraparesis with normal neuroimaging, a progressive spastic dystonic motor disorder, and adult-onset psychological difficulties with intracranial calcification. Homozygous missense mutations were recorded in five families. We observed a p.Pro193Ala variant in the heterozygous state in 22 of 23 families with compound heterozygous mutations. We also ascertained 11 cases from nine families with a p.Gly1007Arg dominant-negative mutation, which occurred de novo in four patients, and was inherited in three families in association with marked phenotypic variability. In 50 of 52 samples from 34 patients, we identified a marked upregulation of type I interferon-stimulated gene transcripts in peripheral blood, with a median interferon score of 16.99 (interquartile range [IQR]: 10.64-25.71) compared with controls (median: 0.93, IQR: 0.57-1.30). Thus, mutations in ADAR1 are associated with a variety of clinically distinct neurological phenotypes presenting from early infancy to adulthood, inherited either as an autosomal recessive or dominant trait. Testing for an interferon signature in blood represents a useful biomarker in this context.

Mutations in SLC12A5 in epilepsy of infancy with migrating focal seizures.

The potassium-chloride co-transporter KCC2, encoded by SLC12A5, plays a fundamental role in fast synaptic inhibition by maintaining a hyperpolarizing gradient for chloride ions. KCC2 dysfunction has been implicated in human epilepsy, but to date, no monogenic KCC2-related epilepsy disorders have been described. Here we show recessive loss-of-function SLC12A5 mutations in patients with a severe infantile-onset pharmacoresistant epilepsy syndrome, epilepsy of infancy with migrating focal seizures (EIMFS). Decreased KCC2 surface expression, reduced protein glycosylation and impaired chloride extrusion contribute to loss of KCC2 activity, thereby impairing normal synaptic inhibition and promoting neuronal excitability in this early-onset epileptic encephalopathy.

Epilepsy due to PNPO mutations: genotype, environment and treatment affect presentation and outcome.

The first described patients with pyridox(am)ine 5'-phosphate oxidase deficiency all had neonatal onset seizures that did not respond to treatment with pyridoxine but responded to treatment with pyridoxal 5'-phosphate. Our data suggest, however, that the clinical spectrum of pyridox(am)ine 5'-phosphate oxidase deficiency is much broader than has been reported in the literature. Sequencing of the PNPO gene was undertaken for a cohort of 82 individuals who had shown a reduction in frequency and severity of seizures in response to pyridoxine or pyridoxal 5'-phosphate. Novel sequence changes were studied using a new cell-free expression system and a mass spectrometry-based assay for pyridoxamine phosphate oxidase. Three groups of patients with PNPO mutations that had reduced enzyme activity were identified: (i) patients with neonatal onset seizures responding to pyridoxal 5'-phosphate (n = 6); (ii) a patient with infantile spasms (onset 5 months) responsive to pyridoxal 5'-phosphate (n = 1); and (iii) patients with seizures starting under 3 months of age responding to pyridoxine (n = 8). Data suggest that certain genotypes (R225H/C and D33V) are more likely to result in seizures that to respond to treatment with pyridoxine. Other mutations seem to be associated with infertility, miscarriage and prematurity. However, the situation is clearly complex with the same combination of mutations being seen in patients who responded and did not respond to pyridoxine. It is possible that pyridoxine responsiveness in PNPO deficiency is affected by prematurity and age at the time of the therapeutic trial. Other additional factors that are likely to influence treatment response and outcome include riboflavin status and how well the foetus has been supplied with vitamin B6 by the mother. For some patients there was a worsening of symptoms on changing from pyridoxine to pyridoxal 5'-phosphate. Many of the mutations in PNPO affected residues involved in binding flavin mononucleotide or pyridoxal 5'-phosphate and many of them showed residual enzyme activity. One sequence change (R116Q), predicted to affect flavin mononucleotide binding and binding of the two PNPO dimers, and with high residual activity was found in Groups (ii) and (iii). This sequence change has been reported in the 1000 Genomes project suggesting it could be a polymorphism but alternatively it could be a common mutation, perhaps responsible for the susceptibility locus for genetic generalized epilepsy on 17q21.32 (close to rs72823592). We believe the reduction in PNPO activity and B6-responsive epilepsy in the patients reported here indicates that it contributes to the pathogenesis of epilepsy.

A de novo gain-of-function mutation in SCN11A causes loss of pain perception.

The sensation of pain protects the body from serious injury. Using exome sequencing, we identified a specific de novo missense mutation in SCN11A in individuals with the congenital inability to experience pain who suffer from recurrent tissue damage and severe mutilations. Heterozygous knock-in mice carrying the orthologous mutation showed reduced sensitivity to pain and self-inflicted tissue lesions, recapitulating aspects of the human phenotype. SCN11A encodes Nav1.9, a voltage-gated sodium ion channel that is primarily expressed in nociceptors, which function as key relay stations for the electrical transmission of pain signals from the periphery to the central nervous system. Mutant Nav1.9 channels displayed excessive activity at resting voltages, causing sustained depolarization of nociceptors, impaired generation of action potentials and aberrant synaptic transmission. The gain-of-function mechanism that underlies this channelopathy suggests an alternative way to modulate pain perception.

Dolichol kinase deficiency (DOLK-CDG) with a purely neurological presentation caused by a novel mutation.

A 4-month old boy presented with multiple epileptic seizure types including West syndrome. Screening for infectious and structural etiologies showed normal results. A metabolic investigation was undertaken to investigate the cause of his neurological disease. Screening for congenital disorders of glycosylation (CDG) by HPLC analysis of serum carbohydrate-deficient transferrin (CDT) showed a type 1 pattern with 18% disialotransferrin (reference < 2%) and 2% asialotransferrin (reference 0). An undiagnosed 10-year old sister with a similar clinical history with infantile spasms at age 4 months, intellectual disability and an autism spectrum disorder, also showed a type 1 CDT pattern. Both siblings lacked dysmorphic features and extra-cerebral symptoms. The boy had cytotoxic edema of the thalamus and mesencephalon on MRI at age 7 months, whereas the girl had normal MRI at age 8 months. Phosphomannomutase (PMM) and phosphomannose isomerase (MPI) activities in cultured fibroblasts were normal, excluding PMM2-CDG and MPI-CDG. Fibroblast lipid-linked oligosaccharide analysis was also normal, suggesting an early defect in glycan assembly. Sequence analysis of the dolichol kinase gene revealed a homozygous new missense mutation (p.M1?; c.2 T > C) in both siblings. In conclusion, two siblings were demonstrated to suffer from DOLK-CDG (MIM 610768) and to be homozygous for a new mutation. They presented with West syndrome and so far show a purely neurological phenotype.