Distinct epilepsy phenotypes and response to drugs in KCNA1 gain- and loss-of function variants.

A wide phenotypic spectrum of neurological diseases is associated with KCNA1 (Kv1.1) variants. To investigate the molecular basis of such a heterogeneous clinical presentation and identify the possible correlation with in vitro phenotypes, we compared the functional consequences of three heterozygous de novo variants (p.P403S, p.P405L, and p.P405S) in Kv1.1 pore region found in four patients with severe developmental and epileptic encephalopathy (DEE), with those of a de novo variant in the voltage sensor (p.A261T) identified in two patients with mild, carbamazepine-responsive, focal epilepsy. Patch-clamp electrophysiology was used to investigate the functional properties of mutant Kv1.1 subunits, both expressed as homomers and heteromers with wild-type Kv1.1 subunits. KCNA1 pore mutations markedly decreased (p. P405S) or fully suppressed (p. P403S, p. P405L) Kv1.1-mediated currents, exerting loss-of-function (LoF) effects. By contrast, channels carrying the p.A261T variant exhibited a hyperpolarizing shift of the activation process, consistent with a gain-of-function (GoF) effect. The present results unveil a novel correlation between in vitro phenotype (GoF vs LoF) and clinical course (mild vs severe) in KCNA1-related phenotypes. The excellent clinical response to carbamazepine observed in the patients carrying the A261T variant suggests an exquisite sensitivity of KCNA1 GoF to sodium channel inhibition that should be further explored.

Diagnostic implications of genetic copy number variation in epilepsy plus.

OBJECTIVE: Copy number variations (CNVs) represent a significant genetic risk for several neurodevelopmental disorders including epilepsy. As knowledge increases, reanalysis of existing data is essential. Reliable estimates of the contribution of CNVs to epilepsies from sizeable populations are not available. METHODS: We assembled a cohort of 1255 patients with preexisting array comparative genomic hybridization or single nucleotide polymorphism array based CNV data. All patients had "epilepsy plus," defined as epilepsy with comorbid features, including intellectual disability, psychiatric symptoms, and other neurological and nonneurological features. CNV classification was conducted using a systematic filtering workflow adapted to epilepsy. RESULTS: Of 1097 patients remaining after genetic data quality control, 120 individuals (10.9%) carried at least one autosomal CNV classified as pathogenic; 19 individuals (1.7%) carried at least one autosomal CNV classified as possibly pathogenic. Eleven patients (1%) carried more than one (possibly) pathogenic CNV. We identified CNVs covering recently reported (HNRNPU) or emerging (RORB) epilepsy genes, and further delineated the phenotype associated with mutations of these genes. Additional novel epilepsy candidate genes emerge from our study. Comparing phenotypic features of pathogenic CNV carriers to those of noncarriers of pathogenic CNVs, we show that patients with nonneurological comorbidities, especially dysmorphism, were more likely to carry pathogenic CNVs (odds ratio = 4.09, confidence interval = 2.51-6.68; P = 2.34 × 10-9 ). Meta-analysis including data from published control groups showed that the presence or absence of epilepsy did not affect the detected frequency of CNVs. SIGNIFICANCE: The use of a specifically adapted workflow enabled identification of pathogenic autosomal CNVs in 10.9% of patients with epilepsy plus, which rose to 12.7% when we also considered possibly pathogenic CNVs. Our data indicate that epilepsy with comorbid features should be considered an indication for patients to be selected for a diagnostic algorithm including CNV detection. Collaborative large-scale CNV reanalysis leads to novel declaration of pathogenicity in unexplained cases and can promote discovery of promising candidate epilepsy genes.

De novo KCNA1 variants in the PVP motif cause infantile epileptic encephalopathy and cognitive impairment similar to recurrent KCNA2 variants.

Derangements in voltage-gated potassium channel function are responsible for a range of paroxysmal neurologic disorders. Pathogenic variants in the KCNA1 gene, which encodes the voltage-gated potassium channel Kv1.1, are responsible for Episodic Ataxia Type 1 (EA1). Patients with EA1 have an increased incidence of epilepsy, but KCNA1 variants have not been described in epileptic encephalopathy. Here, we describe four patients with infantile-onset epilepsy and cognitive impairment who harbor de novo KCNA1 variants located within the Kv-specific Pro-Val-Pro (PVP) motif which is essential for channel gating. The first two patients have KCNA1 variants resulting in (p.Pro405Ser) and (p.Pro405Leu), respectively, and a set of identical twins has a variant affecting a nearby residue (p.Pro403Ser). Notably, recurrent de novo variants in the paralogous PVP motif of KCNA2 have previously been shown to abolish channel function and also cause early-onset epileptic encephalopathy. Importantly, this report extends the range of phenotypes associated with KCNA1 variants to include epileptic encephalopathy when the PVP motif is involved.

Diagnostic Targeted Resequencing in 349 Patients with Drug-Resistant Pediatric Epilepsies Identifies Causative Mutations in 30 Different Genes.

Targeted resequencing gene panels are used in the diagnostic setting to identify gene defects in epilepsy. We performed targeted resequencing using a 30-genes panel and a 95-genes panel in 349 patients with drug-resistant epilepsies beginning in the first years of life. We identified 71 pathogenic variants, 42 of which novel, in 30 genes, corresponding to 20.3% of the probands. In 66% of mutation positive patients, epilepsy onset occurred before the age of 6 months. The 95-genes panel allowed a genetic diagnosis in 22 (6.3%) patients that would have otherwise been missed using the 30-gene panel. About 50% of mutations were identified in genes coding for sodium and potassium channel components. SCN2A was the most frequently mutated gene followed by SCN1A, KCNQ2, STXBP1, SCN8A, CDKL5, and MECP2. Twenty-nine mutations were identified in 23 additional genes, most of them recently associated with epilepsy. Our data show that panels targeting about 100 genes represent the best cost-effective diagnostic option in pediatric drug-resistant epilepsies. They enable molecular diagnosis of atypical phenotypes, allowing to broaden phenotype-genotype correlations. Molecular diagnosis might influence patients' management and translate into better and specific treatment recommendations in some conditions.

Recessive mutations in SLC35A3 cause early onset epileptic encephalopathy with skeletal defects.

We describe the clinical and whole genome sequencing (WGS) study of a non-consanguineous Italian family in which two siblings, a boy and a girl, manifesting a severe epileptic encephalopathy (EE) with skeletal abnormalities, carried novel SLC35A3 compound heterozygous mutations. Both siblings exhibited infantile spasms, associated with focal, and tonic vibratory seizures from early infancy. EEG recordings showed a suppression-burst (SB) pattern and multifocal paroxysmal activity in both. In addition both had quadriplegia, acquired microcephaly, and severe intellectual disability. General examination showed distal arthrogryposis predominant in the hands in both siblings and severe left dorso-lumbar convex scoliosis in one. WGS of the siblings-parents quartet identified novel compound heterozygous mutations in SLC35A3 in both children. SLC35A3 encodes the major Golgi uridine diphosphate N-acetylglucosamine transporter. With this study, we add SLC35A3 to the gene list of epilepsies. Neurological symptoms and skeletal abnormalities might result from impaired glycosylation of proteins involved in normal development and function of the central nervous system and skeletal apparatus.

The hyperkinetic movement disorder of FOXG1-related epileptic-dyskinetic encephalopathy.

AIM: Forkhead Box G1 (FOXG1) syndrome is a developmental encephalopathy characterized by postnatal microcephaly, structural brain abnormalities, facial dysmorphisms, severe delay with absent language, defective social interactions, and epilepsy. Abnormal movements in FOXG1 syndrome have often been mentioned but not characterized. METHOD: We clinically assessed and analysed video recordings of eight patients with different mutations or copy number variations affecting the FOXG1 gene and describe the peculiar pattern of the associated movement disorder. RESULTS: The age of the patients in the study ranged from 2 to 17 years old (six females, two males). They had severe epilepsy and exhibited a complex motor disorder including various combinations of dyskinetic and hyperkinetic movements featuring dystonia, chorea, and athetosis. The onset of the movement disorder was apparent within the first year of life, reached its maximum expression within months, and then remained stable. INTERPRETATION: A hyperkinetic-dyskinetic movement disorder emerges as a distinctive feature of the FOXG1-related phenotype. FOXG1 syndrome is as an epileptic-dyskinetic encephalopathy whose clinical presentation bears similarities with ARX- and STXBP1-gene related encephalopathies.

Recurrent drop attacks in early childhood as presenting symptom of benign hereditary chorea caused by TITF1 gene mutations.

Drop attacks are sudden, spontaneous falls without loss of consciousness, followed by rapid recovery. Causes in children include severe epilepsies, movement disorders, cataplexy, and psychiatric disorders. We describe two children (a 3-year-old female and a 12-year-old male) with mild neuromotor delay and sudden falls appearing upon starting to walk. Extensive clinical and laboratory investigation was unremarkable. Twenty to 22 months after the onset of falls, both children developed subtle choreiform movements, affecting all four limbs, leading to frequent falls, at times causing traumatic injury. A heterozygous mutation of the TITF1/NKX2-1 gene (14q13) was detected in both patients, allowing the diagnosis of benign hereditary chorea (BHC). Treatment with levodopa attenuated abnormal movements and led to disappearance of drop attacks. A diagnosis of BHC should be considered in young children with recurrent and unexplained drop attacks, especially if associated with neuromotor delay, even in the absence of choreiform movements.

Factors of risk and maintenance for eating disorders: psychometric exploration of the cross-cultural questionnaire (CCQ) across five European countries.

OBJECTIVE: To explore the psychometric properties of the Cross-Cultural Questionnaire (CCQ), a new self-report tool for assessing factors of risk and maintenance for eating disorders (ED). METHOD: Data was collected during a multi-centre case-control study. The sample included 854 ED patients and 784 healthy participants from the UK, Spain, Austria, Slovenia and Italy. Exploratory factor analyses examined the factor structure of each section of the CCQ, and Cronbach's alpha coefficients valued the internal consistency of each derived scale score. Logistic regression and receiver operating characteristic (ROC) curve procedure assessed the screening accuracy and predictive validity of the empirical factors. RESULTS: Based on a total of 127 items, nine dimensions emerged, with satisfactory internal consistency and high congruence between countries. CCQ scores demonstrated satisfactory accuracy for discriminating between ED cases and controls (area under the ROC curve = 0.88). Most of the items achieved discriminative accuracy. CONCLUSIONS: This study offers preliminary evidence that the CCQ, available in five languages, is a useful and valid tool to assess factors of risk and maintenance for EDs.

Whole Exome Sequencing Is the Minimal Technological Approach in Probands Born to Consanguineous Couples.

We report on two siblings suffering from different pathogenic conditions, born to consanguineous parents. A multigene panel for brain malformations and microcephaly identified the homozygous splicing variant NM_005886.3:c.1416+1del in the KATNB1 gene in the older sister. On the other hand, exome sequencing revealed the homozygous frameshift variant NM_005245.4:c.9729del in the FAT1 gene in the younger sister, who had a more complex phenotype: in addition to bilateral anophthalmia and heart defects, she showed a right split foot with 4 toes, 5 metacarpals, second toe duplication and preaxial polydactyly on the right hand. These features have been never reported before in patients with pathogenic FAT1 variants and support the role of this gene in the development of limb buds. Notably, each parent was heterozygous for both of these variants, which were ultra-rare and rare, respectively. This study raises awareness about the value of using whole exome/genome sequencing rather than targeted gene panels when testing affected offspring born to consanguineous couples. In this way, exomic data from the parents are also made available for carrier screening, to identify heterozygous pathogenetic and likely pathogenetic variants in genes responsible for other recessive conditions, which may pose a risk for subsequent pregnancies.

Association of NTRK3 and its interaction with NGF suggest an altered cross-regulation of the neurotrophin signaling pathway in eating disorders.

Eating disorders (EDs) are complex psychiatric diseases that include anorexia nervosa and bulimia nervosa, and have higher than 50% heritability. Previous studies have found association of BDNF and NTRK2 to ED, while animal models suggest that other neurotrophin genes might also be involved in eating behavior. We have performed a family-based association study with 151 TagSNPs covering 10 neurotrophin signaling genes: NGFB, BDNF, NTRK1, NGFR/p75, NTF4/5, NTRK2, NTF3, NTRK3, CNTF and CNTFR in 371 ED trios of Spanish, French and German origin. Besides several nominal associations, we found a strong significant association after correcting for multiple testing (P = 1.04 x 10(-4)) between ED and rs7180942, located in the NTRK3 gene, which followed an overdominant model of inheritance. Interestingly, HapMap unrelated individuals carrying the rs7180942 risk genotypes for ED showed higher levels of expression of NTRK3 in lymphoblastoid cell lines. Furthermore, higher expression of the orthologous murine Ntrk3 gene was also detected in the hypothalamus of the anx/anx mouse model of anorexia. Finally, variants in NGFB gene appear to modify the risk conferred by the NTRK3 rs7180942 risk genotypes (P = 4.0 x 10(-5)) showing a synergistic epistatic interaction. The reported data, in addition to the previous reported findings for BDNF and NTRK2, point neurotrophin signaling genes as key regulators of eating behavior and their altered cross-regulation as susceptibility factors for EDs.

Variable epilepsy phenotypes associated with a familial intragenic deletion of the SCN1A gene.

Deletions and duplications/amplifications of the α1-sodium channel subunit (SCN1A) gene occur in about 12% of patients with Dravet syndrome (DS) who are otherwise mutation-negative. Such genomic abnormalities cause loss of function, with severe phenotypes, reproductive disadvantage and, therefore, sporadic occurrence. Inherited mutations, occurring in ∼5% of patients with DS, are usually missense; transmission occurs from a mildly affected parent exhibiting febrile seizures (FS) or the generalized epilepsy with febrile seizures plus (GEFS+) spectrum. We identified an intragenic SCN1A deletion in a three-generation, clinically heterogeneous family. Sequence analysis of SCN9A, a putative modifier, ruled out pathogenic mutations, variants, or putative disease-associated haplotype segregating with phenotype severity. Intrafamilial variability in phenotype severity indicates that SCN1A loss of function causes a phenotypic spectrum in which seizures precipitated by fever are prominent and schematic syndrome subdivisions would be inappropriate. SCN1A deletions should be ruled out even in individuals with mild phenotypes.

Implication of GAB2 gene polymorphism in Italian patients with Alzheimer's disease.

A common polymorphism (rs2373115) in the GRB-associated binding protein 2 (GAB2) gene has been recently associated with the risk of developing Alzheimer's disease (AD) in 644 apolipoprotein E (ApoE) epsilon4 carriers. In order to assess the involvement of the GAB2 polymorphism in the risk of developing AD, we analyzed the genotype and allele distributions of the GAB2 rs2373115 polymorphism in 579 Italian subjects. Our results support a possible implication of GAB2 genetic variant in AD. However, the observed association was confined to ApoE epsilon4 non-carriers, thus suggesting a possible role of GAB2 as an independent risk factor for AD.

Associations of individual and family eating patterns during childhood and early adolescence: a multicentre European study of associated eating disorder factors.

The objective of this study was to examine whether there is an association between individual and family eating patterns during childhood and early adolescence and the likelihood of developing a subsequent eating disorder (ED). A total of 1664 participants took part in the study. The ED cases (n 879) were referred for assessment and treatment to specialized ED units in five different European countries and were compared to a control group of healthy individuals (n 785). Participants completed the Early Eating Environmental Subscale of the Cross-Cultural (Environmental) Questionnaire, a retrospective measure, which has been developed as part of a European multicentre trial in order to detect dimensions associated with ED in different countries. In the control group, also the General Health Questionnaire-28 (GHQ-28), the semi-structured clinical interview (SCID-I) and the Eating Attitudes Test (EAT-26) were used. Five individually Categorical Principal Components Analysis (CatPCA) procedures were adjusted, one for each theoretically expected factor. Logistic regression analyses indicated that the domains with the strongest effects from the CatPCA scores in the total sample were: food used as individualization, and control and rules about food. On the other hand, healthy eating was negatively related to a subsequent ED. When differences between countries were assessed, results indicated that the pattern of associated ED factors did vary between countries. There was very little difference in early eating behaviour on the subtypes of ED. These findings suggest that the fragmentation of meals within the family and an excessive importance given to food by the individual and the family are linked to the later development of an ED.

Multiple genomic copy number variants associated with periventricular nodular heterotopia indicate extreme genetic heterogeneity.

Periventricular nodular heterotopia (PNH) is a brain malformation in which nodules of neurons are ectopically retained along the lateral ventricles. Genetic causes include FLNA abnormalities (classical X-linked PNH), rare variants in ARFGEF2, DCHS1, ERMARD, FAT4, INTS8, MAP1B, MCPH1, and NEDD4L, as well as several chromosomal abnormalities. We performed array-CGH in 106 patients with different malformations of cortical development (MCD) and looked for common pathways possibly involved in PNH. Forty-two patients, including two parent/proband couples, exhibited PNH associated or not with other brain abnormalities, 44 had polymicrogyria and 20 had rarer MCDs. We found an enrichment of either large rearrangements or cryptic copy number variants (CNVs) in PNH (15/42, 35.7%) vs polymicrogyria (4/44, 9.1%) (i.e., 5.6 times increased risk for PNH of carrying a pathogenic CNV). CNVs in seven genomic regions (2p11.2q12.1, 4p15, 14q11.2q12, 16p13.3, 19q13.33, 20q13.33, 22q11) represented novel, potentially causative, associations with PNH. Through in silico analysis of genes included in imbalances whose breakpoints were clearly detailed, we detected in 9/12 unrelated patients in our series and in 15/24 previously published patients, a significant (P < 0.05) overrepresentation of genes involved in vesicle-mediated transport. Rare genomic imbalances, either small CNVs or large rearrangements, are cumulatively a frequent cause of PNH. Dysregulation of specific cellular mechanisms might play a key pathogenic role in PNH but it remains to be determined whether this is exerted through single genes or the cumulative dosage effect of more genes. Array-CGH should be considered as a first-line diagnostic test in PNH, especially if sporadic and non-classical.

Lack of association between TNF-alpha polymorphisms and Alzheimer's disease in an Italian cohort.

Tumor necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine that plays an important role in the inflammatory process that can be observed in Alzheimer's disease (AD) brain. Different functional promoter polymorphisms within genes modulating inflammation have been demonstrated to elevate the AD risk; thus, we studied five common variations within the promoter region of the TNF-alpha gene in 609 subjects (253 AD patients and 356 controls). No positive associations were found, confirming the greater part of previous studies. Moreover, we also investigated the combined haplotypes of the five different polymorphisms without finding a positive association. Thus, the present investigation does not support the proposal that common nucleotide variations in the TNF-alpha gene can influence the development of AD at least in Italian population.

KIBRA gene variants are associated with episodic memory performance in subjective memory complaints.

The KIBRA gene encodes a cytoplasmatic protein, a member of the signal transduction protein family, expressed mainly in the brain. Recent studies have implicated the involvement of a genetic variation in the KIBRA gene (T allele) in human memory in normal subjects and in the risk of developing Alzheimer's disease (AD). We report here the distribution of the KIBRA genetic variant and the Apolipoprotein E (ApoE) epsilon4 allele and their association with neuropsychological measures in older adults reporting problems with everyday memory (subjective memory complaints, SMC). We found that SMC subjects with the CT/TT genotype performed more poorly than those with the CC genotype on long-term memory tests. Thus, in our opinion, these data suggest that the KIBRA genotype could affect memory performance in a different way in those that complain of memory deficits compared to those that do not.

Present and lifetime comorbidity of tobacco, alcohol and drug use in eating disorders: a European multicenter study.

OBJECTIVES: To assess the differences in comorbid lifetime and current substance use (tobacco, alcohol and drug use) between eating disorder (ED) patients and healthy controls in five different European countries. METHOD: A total of 1664 participants took part in the present study. ED cases (n=879) were referred to specialized ED units in five European countries. The ED cases were compared to a balanced control group of 785 healthy individuals. ASSESSMENT: Participants completed the Substance Use Subscale of the Cross Cultural (Environmental) Questionnaire (CCQ), a measure of lifetime tobacco, alcohol and drug use. In the control group, also the GHQ-28, the SCID-I interview and the EAT-26 were used. RESULTS: ED patients had higher lifetime and current tobacco and general drug use. The only non-significant result was obtained for lifetime and current alcohol use. Significant differences across ED subdiagnoses and controls also emerged, with BN and AN-BP generally presenting the highest and AN-R and controls the lowest rates. The only exception was detected for alcohol use where EDNOS demonstrated the highest values. Only a few cultural differences between countries emerged. CONCLUSIONS: With the exception of alcohol consumption, tobacco and drug use appear to be more prevalent in ED patients than healthy controls. The differential risk observed in patients with bulimic features might be related to differences in temperament or might be the result of increased sensitivity to reward.

Mutational screening analysis of DHCR24/seladin-1 gene in Italian familial Alzheimer's disease.

There is evidence that both environmental and genetic factors may play a role in the pathogenesis of Alzheimer's disease (AD). The amount of brain cholesterol, for instance, has been suggested to play a role in the development of the disease. Accordingly, the Apolipoprotein E (ApoE) epsilon4 allele has been identified as a major risk factor for the occurrence of AD. The product of the DHCR24/seladin-1 gene has enzymatic activity, which converts desmosterol into cholesterol. The expression of this gene, which confers protection against beta-amyloid toxicity and from oxidative stress, is downregulated in AD vulnerable brain regions and it has been proposed as possibly involved in the pathogenesis of this disease. In this study, we evaluated the possible genetic contribution of the DHCR24/seladin-1 gene to Italian familial cases of AD. The exons 1-9 of this gene from 100 patients were subjected to mutation screening analysis. We identified a new C to T transition in exon 1 (Leu60Leu) and a previously described C to T transition in exon 7 (Ile342Ile-rs718265). Our preliminary results suggest the absence of an association between DHCR24/seladin-1 genotypes and AD in the Italian population.