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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.

Carcinoma in situ (CIS) is the common precursor of histologically heterogeneous testicular germ cell tumors (TGCTs), which in recent decades have markedly increased and now are the most common malignancy of young men. Using genome-wide gene expression profiling, we identified >200 genes highly expressed in testicular CIS, including many never reported in testicular neoplasms. Expression was further verified by semiquantitative reverse transcription-PCR and in situ hybridization. Among the highest expressed genes were NANOG and POU5F1, and reverse transcription-PCR revealed possible changes in their stoichiometry on progression into embryonic carcinoma. We compared the CIS expression profile with patterns reported in embryonic stem cells (ESCs), which revealed a substantial overlap that may be as high as 50%. We also demonstrated an over-representation of expressed genes in regions of 17q and 12, reported as unstable in cultured ESCs. The close similarity between CIS and ESCs explains the pluripotency of CIS. Moreover, the findings are consistent with an early prenatal origin of TGCTs and thus suggest that etiologic factors operating in utero are of primary importance for the incidence trends of TGCTs. Finally, some of the highly expressed genes identified in this study are promising candidates for new diagnostic markers for CIS and/or TGCTs.

Genes found up-regulated in testicular samples with CIS (fold change)

Supplementary Figure 2 from Embryonic Stem Cell-Like Features of Testicular Carcinoma in Situ Revealed by Genome-Wide Gene Expression Profiling

Supplementary Figure 1 from Embryonic Stem Cell-Like Features of Testicular Carcinoma in Situ Revealed by Genome-Wide Gene Expression Profiling

Background: Preterm infants have an increased risk of neurodevelopmental disorders. We established a direct quantitative comparison of the association between the degree of prematurity and three different neurodevelopmental disorders. Methods: In this cohort study, we combined data from 995,498 children in the Danish Medical Birth Register, from birth years 1997–2013, with information on cerebral palsy, epilepsy, and special educational needs. We estimated the gestational week-specific prevalence and risk for each of the disorders. Results: The risk ratio of cerebral palsy at gestational weeks 21–24, compared to term birth, was more than ten times higher than for the two other disorders. The prevalence of epilepsy and special educational needs declined almost parallel, with 9.2% (4.6%–13.5%) and 12.5% (11.2%–13.7%), respectively, per week of gestation toward term birth. Cerebral palsy did not decline similarly: from gestational weeks 21–24 until week 29 the prevalence declined insignificantly by 0.6% (−11.1%–11.0%) per week; whereas from week 29 until term, the prevalence declined markedly by 36.7% (25.9%–45.9%) per week. Conclusions: The prevalence and risk of cerebral palsy are affected differently by the degree of prematurity compared with epilepsy and special educational needs, possibly reflecting important differences in cerebral pathophysiology. Impact: For each week of gestation toward term birth, there was a clear log-linear decline in the prevalence of early childhood epilepsy and special educational needs.In contrast, the risk of cerebral palsy was high at the earliest gestational age, and the prevalence did not decline significantly until gestational week 29, from where it declined notably by nearly 40% for each week of gestation until term birth.Our results indicate important differences in the pathophysiological processes that associate preterm birth with these three neurodevelopmental disorders.

This review finds that, in children and adults with epilepsy, there are several treatment options. Multiple antiseizure medications are available and in case of drug-resistant epilepsy, a non-pharmacological approach is recommended, including epilepsy surgery, vagus nerve stimulation, or ketogenic diet treatment. The aim of the treatment is to avoid further seizures, but also to avoid negative cognitive, psychological, and social consequences of epilepsy.

The recurrent seizures of pediatric drug-resistant epilepsy (DRE) are known to impair brain development and can lead to a loss in cognitive functioning. Surgery is increasingly being used to treat children with DRE. This study investigates the pre- and postoperative cognitive function in a pediatric epilepsy surgery cohort as well as predictive determinants of change in intelligence quotient (IQ) following surgery.A consecutive series of 91 Danish children who underwent focal resective epilepsy surgery between January 1996 and December 2016 were included. All underwent preoperative cognitive evaluation and were reevaluated at 1-year and/or 2-year follow-up. Single-operated and multi-operated patients were examined separately.79 of 91 patients were single-operated. Single-operated patients received less anti-epileptic drugs (AED) and experienced a decrease in seizure frequency postoperatively, p0.001. IQ increased postoperatively (IQ change ± standard deviation: 3.3 ± 14.0), p0.05. High preoperative seizure frequency was a significant predictor for decreased IQ, p0.01. Multi-operated patients did not experience a reduction in AED treatment. Surgery and continued AED treatment did, however, result in significantly better seizure control, p0.01. IQ remained unchanged in multi-operated patients.Epilepsy surgery allowed for IQ gains in single-operated patients. Preoperative seizure frequency was a significant predictor of IQ change following surgery. Interactions between other, not included, possible predictors remain to be examined. Single-operated patients had the best cognitive outcome. The inclusion of a non-surgical control group is needed to assess the extent of the beneficial effects of surgery on cognitive ability.

Background: The use of homemade tube feeding formula has become increasingly popular for children requiring enteral nutrition. This project aimed to investigate nutrition and preparation of blenderized tube feeding in the field of children and adolescents with neurological impairment.Methods: A scoping review was performed using established methodologies. In January 2021, we searched PubMed, Embase, CINAHL Complete, the Cochrane Central Register of Controlled Trials, and gray literature to identify relevant articles.Major findings: Twenty-two papers were included describing the composition of food items, preparation procedures, and food safety. No randomized controlled trials and only a few prospective studies were included. A broad variety of food items from all food groups and many examples of recipes were presented. Most recipes provided 1.0 kcal/ml but tended to contain less energy and nutrients than expected, which could be due to preparation issues, such as sieving and the high viscosity of the blend. Preparation requires a commercial-grade household blender and diligence to ensure thorough household hygiene for adequate food safety.Conclusions: This review revealed practical experience in the nutrition and preparation aspects of blenderized tube feeding but minimal empirical evidence. Multiple examples of the composition of food items and preparation procedures for blenderized tube feeding were found, but uncertainty regarding the ideal composition or preparation was also exposed. The future of blenderized tube feeding would benefit from clinically tested recipes that include an evaluation of nutrients, viscosity, and microbial contamination, as well as the effect of the food's appearance and scent on the target group.

Aim: Children with dyskinetic cerebral palsy (CP) are often severely affected and effective treatment is difficult, due to different underlying disease mechanisms. Comprehensive systematic movement disorder evaluations were carried out on patients with this disorder. Methods: Patients born from 1995 to 2007 were identified from the Danish Cerebral Palsy Register and referrals to the neuropaediatric centre, Rigshospitalet, Copenhagen. They were classified by gross motor function, manual functional ability, communication ability, dystonia and spasticity. Electromyography was carried out on the upper and lower limbs. Magnetic resonance imaging scans were revised, and aetiological searches for underlying genetic disorders were performed. Results: We investigated 25 patients with dyskinetic CP at a mean age of 11.7 years. Dystonia, spasticity and rigidity were found in the upper limbs of 21, four and six children, respectively, and in the lower limbs of 18, 18 and three children. The mean total Burke-Fahn-Marsden score for dystonia was 45.02, and the mean Disability Impairment Scale level was 38% for dystonia and 13% for choreoathetosis. Sustained electromyography activity was observed in 20/25 children. Stretching increased electromyography activity more in children with spasticity. There were 10 re-classifications. Conclusion: The children had heterogenic characteristics, and 40% were reclassified after systematic movement disorder evaluation.

AIM: To investigate reasons for the declining prevalence of cerebral palsy (CP) in children born at term in Denmark by evaluating obstetric and neonatal factors associated with CP, and their changes over time.METHOD: In this cohort study, we included 987 495 children (504 600 [51.1%] males and 482 895 [48.9%] females) born after 37 completed gestational weeks during birth years 1997 to 2013. Risk ratios of CP for each factor were calculated with log-binominal regression analyses. Significant factors were evaluated concerning their development in prevalence over time.RESULTS: In the antenatal period, there were significant associations with an increased risk of CP and high maternal body mass index (BMI), smoking during pregnancy, nulliparity, male sex, gestational age, and low birthweight. In the study period, fewer females smoked during pregnancy and fewer children were born post-term, dropping from 22.6% to 11.4% and 9.4% to 2.5% respectively. Conversely, the proportion of females with high BMI increased. Most significant risk factors were found in the neonatal period, with an increase in children with diagnosed birth defects and children admitted to neonatal care.INTERPRETATION: Reasons for the declining prevalence of CP appear to be multifactorial and likely include the decline in maternal smoking and children born post-term along with centralization and advances in neonatal treatment.

AIM: To analyse trends in prevalence and severity of cerebral palsy (CP) in Denmark in birth years 1999-2007 and compare with previous periods.METHOD: Data has been collected uniformly in the Danish cerebral palsy national register nationwide since 1995. Rates in the time periods 1999-2001, 2002-2004 and 2005-2007 covering 585,393 births were analysed by gestational age and subtypes.RESULTS: Total number of CP cases in the period was 1165. The overall prevalence of CP decreased significantly from 2.1 in 1999-2001 to 1.8 in 2005-2007 per 1000 livebirths (p = 0.022). The decline was only significant for children born at term (p = 0.007) but not for the preterm (p = 0.44). The decline in children born at term was based on a decrease in bilateral spastic CP (n = 117 in years 1999-2001 and n = 59 in 2005-2007). Multidisciplinary obstetric skills training with neonatal resuscitation in Denmark was initiated in 2003 and timely associated with the decrease. The prevalence of unilateral spastic CP the prevalence did not change, but in the two last time periods more children had a right-sided than left-sided unilateral spastic CP.CONCLUSION: The decline in rate of CP seen in 2005-2007 as compared to 1999-2001 was mainly based on fewer cases of severe spastic CP in term infants. We hypothesize that improved neonatal resuscitation in the delivery room may be partly responsible for the decrease. In premature children the decline was not significant in this time period, but has been dramatically decreasing in the years before the time period here analysed.

We report detailed functional analyses and genotype-phenotype correlations in 433 individuals carrying disease-causing variants in SCN8A, encoding the voltage-gated Na+ channel NaV1.6. Five different clinical subgroups could be identified: 1) Benign familial infantile epilepsy (BFIE) (n=17, normal cognition, treatable seizures), 2) intermediate epilepsy (n=36, mild ID, partially pharmacoresponsive), 3) developmental and epileptic encephalopathy (DEE, n=191, severe ID, majority pharmacoresistant), 4) generalized epilepsy (n=21, mild to moderate ID, frequently with absence seizures), and 5) affected individuals without epilepsy (n=25, mild to moderate ID). Groups 1-3 presented with early-onset (median: four months) focal or multifocal seizures and epileptic discharges, whereas the onset of seizures in group 4 was later (median: 39 months) with generalized epileptic discharges. The epilepsy was not classifiable in 143 individuals. 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 DEE showed a strong gain-of-function (GOF, hyperpolarising shift of steady-state activation, strongly increased neuronal firing rate), and one variant causing BFIE or intermediate epilepsy showed a mild GOF (defective fast inactivation, less increased firing). In contrast, all three variants causing generalized epilepsy induced a loss-of-function (LOF, reduced current amplitudes, depolarising shift of steady-state activation, reduced neuronal firing). Including previous studies, functional effects were known for 165 individuals. All 133 individuals carrying GOF variants had either focal (76, groups 1-3), or unclassifiable epilepsy (37), whereas 32 with LOF variants had either generalized (14), no (11) or unclassifiable (5) epilepsy; only two had DEE. Computational modeling in the GOF group revealed a significant correlation between the severity of the electrophysiological and clinical phenotypes. GOF variant carriers responded significantly better to sodium channel blockers (SCBs) than to other anti-seizure medications, and the same applied for all individuals of 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 LOF variant carriers and the extent of the electrophysiological dysfunction of the GOF variants is a main determinant of the severity of the clinical phenotype in focal epilepsies. Our pharmacological data indicate that SCBs present a therapeutic treatment option in early onset SCN8A-related focal epilepsy.

Preterm infants have an increased risk of neurodevelopmental disorders. We established a direct quantitative comparison of the association between the degree of prematurity and three different neurodevelopmental disorders.In this cohort study, we combined data from 995,498 children in the Danish Medical Birth Register, from birth years 1997-2013, with information on cerebral palsy, epilepsy, and special educational needs. We estimated the gestational week-specific prevalence and risk for each of the disorders.The risk ratio of cerebral palsy at gestational weeks 21-24, compared to term birth, was more than ten times higher than for the two other disorders. The prevalence of epilepsy and special educational needs declined almost parallel, with 9.2% (4.6%-13.5%) and 12.5% (11.2%-13.7%), respectively, per week of gestation toward term birth. Cerebral palsy did not decline similarly: from gestational weeks 21-24 until week 29 the prevalence declined insignificantly by 0.6% (-11.1%-11.0%) per week; whereas from week 29 until term, the prevalence declined markedly by 36.7% (25.9%-45.9%) per week.The prevalence and risk of cerebral palsy are affected differently by the degree of prematurity compared with epilepsy and special educational needs, possibly reflecting important differences in cerebral pathophysiology.For each week of gestation toward term birth, there was a clear log-linear decline in the prevalence of early childhood epilepsy and special educational needs. In contrast, the risk of cerebral palsy was high at the earliest gestational age, and the prevalence did not decline significantly until gestational week 29, from where it declined notably by nearly 40% for each week of gestation until term birth. Our results indicate important differences in the pathophysiological processes that associate preterm birth with these three neurodevelopmental disorders.

Aim To quantify and analyse the prevalence and clinical features of cerebral palsy (CP) in Denmark for birth years 2008–2013 and compare results with previous periods. Method A nationwide register-based study covering all children with a confirmed diagnosis of CP born in Denmark. Information about CP subtype, aetiology and severity was collected from the Cerebral Palsy Follow-up Program and supplemented from medical files. Data from the Danish Medical Birth Register was included, and the results were compared to previous data from the Danish National Cerebral Palsy Register. Prevalence per 1000 live births and proportions were analysed using the Cochran-Armitage test for trend. Results The period covered 368,618 live births and 636 children with CP, making the overall prevalence for the period 1.73 per 1000 live births. This was significantly lower than the prevalence of 1.99 for the previous period 1999–2007 (p = 0.004). The decline in prevalence between the two periods was mainly due to a decrease in children with bilateral spastic and dyskinetic CP born after 37 gestational weeks. The decline in prevalence was accompanied by a smaller proportion of children with associated impairment. Conclusion We found a decrease in prevalence and severity in CP among Danish children. The decline was most pronounced in children born after 37 gestational weeks with severe subtypes of CP. National guidelines that recommend induction of labour before the completion of week 42 and therapeutic hypothermia for term neonates with hypoxic-ischaemic encephalopathy, may have contributed to the decline.

Ion channel mutations can cause distinct neuropsychiatric diseases. We first studied the biophysical and neurophysiological consequences of four mutations in the human Na+ channel gene SCN8A causing either mild (E1483K) or severe epilepsy (R1872W), or intellectual disability and autism without epilepsy (R1620L, A1622D). Only combined electrophysiological recordings of transfected wild-type or mutant channels in both neuroblastoma cells and primary cultured neurons revealed clear genotype-phenotype correlations. The E1483K mutation causing mild epilepsy showed no significant biophysical changes, whereas the R1872W mutation causing severe epilepsy induced clear gain-of-function biophysical changes in neuroblastoma cells. However, both mutations increased neuronal firing in primary neuronal cultures. In contrast, the R1620L mutation associated with intellectual disability and autism-but not epilepsy-reduced Na+ current density in neuroblastoma cells and expectedly decreased neuronal firing. Interestingly, for the fourth mutation, A1622D, causing severe intellectual disability and autism without epilepsy, we observed a dramatic slowing of fast inactivation in neuroblastoma cells, which induced a depolarization block in neurons with a reduction of neuronal firing. This latter finding was corroborated by computational modelling. In a second series of experiments, we recorded three more mutations (G1475R, M1760I, G964R, causing intermediate or severe epilepsy, or intellectual disability without epilepsy, respectively) that revealed similar results confirming clear genotype-phenotype relationships. We found intermediate or severe gain-of-function biophysical changes and increases in neuronal firing for the two epilepsy-causing mutations and decreased firing for the loss-of-function mutation causing intellectual disability. We conclude that studies in neurons are crucial to understand disease mechanisms, which here indicate that increased or decreased neuronal firing is responsible for distinct clinical phenotypes.

AIM: We aimed at describing clinical findings in children with dyskinetic as compared to bilateral spastic cerebral palsy (CP).METHODS: Data was extracted from the Danish nationwide CP register. Participants were born in 1999-2007 and were 5-6 years at ascertainment.RESULTS: The total number of CP cases was 1,165 of which 92 had dyskinetic and 540 bilateral spastic CP. Prevalence of dyskinetic CP was 0.16 per 1000 live-births. In participants with dyskinetic compared to bilateral spastic CP, there was more frequently an Apgar level less than five at five minutes (22.7% versus 11.2%) and neonatal seizures (43.5% versus 28.5%), but less respiratory deficiency, hyperbilirubinaemia, and sepsis. Impairment based on gross motor function classification was more severe in dyskinetic CP (level III-V 90.0% versus 66.0%). In dyskinetic CP there was a high rate of reduced developmental quotient (68.1%), visual impairment (39.3%), and epilepsy (51.6%). Basal ganglia lesions were more prevalent in dyskinetic compared to bilateral spastic CP (27.7% vs. 12.8%).CONCLUSION: Cases of dyskinetic CP had overlapping clinical features with cases of bilateral spastic CP, but differed significantly in several perinatal risk factors. The children with dyskinetic CP had experienced more peri- or neonatal adverse events, and neurodevelopmental impairment was severe. This article is protected by copyright. All rights reserved.

Infantile spasms is one of the epileptic encephalopathies. These have a variety of established etiological factors, but in a subset of cases, the underlying cause is not evident despite extensive investigations. These cases are referred to as idiopathic or cryptogenic infantile spasms. We report on two cases where the etiology was established by high-resolution array-comparative genomic hybridization as a de novo deletion in the STXBP1 gene. We propose to include high-resolution array-comparative genomic hybridization as a part of the diagnostic evaluation program in patients with infantile spasms with an unknown etiology. Establishment of the etiology has important implications for prognosis and genetic counseling.

AimGerm cell tumours (GCTs) are a rare heterogeneous tumour group derived from primordial germ cells, which can be benign or malignant and occur in the gonads or extragonadally. This study mapped the paediatric GCTs in Denmark from 1984 to 2013 to study the incidence and outcome.MethodsWe identified paediatric GCTs from the Danish Childhood Cancer and National Pathology Registries and reviewed the case records for patient characteristics, tumour characteristics and clinical outcome.ResultsWe identified 403 (71% female) paediatric GCTs and the crude incidence was 1.43 per 100 000. Of these, 79 (20%) were malignant, 39 (10%) were potentially malignant and 285 (70%) were benign. Extragonadal GCTs (39%) were mainly observed in early childhood and were predominately sacrococcygeal teratomas. Gonadal GCTs (61%) in late childhood were most frequently mature teratomas in the ovaries. Nearly all patients underwent surgery. Of the malignant tumours, 62% were treated with chemotherapy. Radiotherapy was only administered to intracranial GCTs. In the cohort, 12 patients died (3%).ConclusionPaediatric GCTs in Denmark were mainly benign and mortality was low, even for malignant tumours. We identified a peak of extragonadal GCTs in early childhood and a peak of gonadal GCTs in late childhood, which was comparable to previous reports.

Purpose Epilepsy surgery is performed based on the assumption that medical refractory epilepsy will continue. Rarely seizure freedom occurs before surgery is performed, while the patient is being evaluated as an epilepsy surgery candidate. The aim of this study was to describe the number of children withdrawn from an epilepsy surgery programme due to unexpected seizure improvement. Methods We retrospectively studied 173 children under 18 years with medical refractory epilepsy referred for epilepsy surgery between 1996 and 2010. Medical records were reviewed in 2012 and 2015. Results At the first evaluation point in 2012, 13 patients were withdrawn from the epilepsy surgery programme due to unexpected marked improvement. In 2015, 6 of them were still seizure free. They had unexpected seizure freedom due to change in AED treatment (n=3) or after a febrile episode (n=3). The mean number of years they had had seizures was 3.4 years (range 0.6–6.2 years) and the number of seizures at inclusion was 209 per month (range 6–750 per month). The duration of follow-up was 6.6 years after inclusion into the epilepsy surgery programme (range 4.0–13.0 years). The aetiology of the epilepsy for these patients was heterotopia (n=1), focal cortical dysplasia (n=3), infarction (n=1) and unknown, with normal MRI (n=1). They all had an IQ in the normal range. Two of the remaining 7 children were operated later. Conclusion Unexpected seizure control may occur during epilepsy surgery evaluation.