Parental mosaicism in epilepsies due to alleged de novo variants.

Severe early onset epilepsies are often caused by de novo pathogenic variants. Few studies have reported the frequency of somatic mosaicism in parents of children with severe epileptic encephalopathies. Here we aim to investigate the frequency of mosaicism in the parents of children with epilepsy caused by alleged de novo variants. We tested parental genomic DNA derived from different tissues for 75 cases using targeted next-generation sequencing. Five parents (6.6%) showed mosaicism at minor allele frequencies of 0.8%-29% for the pathogenic variant detected in their offspring. Parental mosaicism was observed in the following genes: SCN1A, SCN2A, SCN8A, and STXBP1. One of the identified parents had epilepsy himself. Our results show that de novo events can occur already in parental tissue and in some cases can be detected in peripheral blood. Consequently, parents affected by low-grade mosaicism are faced with an increased recurrence risk for transmitting the pathogenic variant, compared to the overall recurrence risk for a second affected child estimated at approximately 1%. However, testing for parental somatic mosaicism will help identifying those parents who truly are at higher risk and will significantly improve genetic counseling in the respective families.

Mutations in GABRB3: From febrile seizures to epileptic encephalopathies.

OBJECTIVE: To examine the role of mutations in GABRB3 encoding the ß3 subunit of the GABAA receptor in individual patients with epilepsy with regard to causality, the spectrum of genetic variants, their pathophysiology, and associated phenotypes. METHODS: We performed massive parallel sequencing of GABRB3 in 416 patients with a range of epileptic encephalopathies and childhood-onset epilepsies and recruited additional patients with epilepsy with GABRB3 mutations from other research and diagnostic programs. RESULTS: We identified 22 patients with heterozygous mutations in GABRB3, including 3 probands from multiplex families. The phenotypic spectrum of the mutation carriers ranged from simple febrile seizures, genetic epilepsies with febrile seizures plus, and epilepsy with myoclonic-atonic seizures to West syndrome and other types of severe, early-onset epileptic encephalopathies. Electrophysiologic analysis of 7 mutations in Xenopus laevis oocytes, using coexpression of wild-type or mutant ß3, together with α5 and γ2s subunits and an automated 2-microelectrode voltage-clamp system, revealed reduced GABA-induced current amplitudes or GABA sensitivity for 5 of 7 mutations. CONCLUSIONS: Our results indicate that GABRB3 mutations are associated with a broad phenotypic spectrum of epilepsies and that reduced receptor function causing GABAergic disinhibition represents the relevant disease mechanism.

Gene Panel Testing in Epileptic Encephalopathies and Familial Epilepsies.

In recent years, several genes have been causally associated with epilepsy. However, making a genetic diagnosis in a patient can still be difficult, since extensive phenotypic and genetic heterogeneity has been observed in many monogenic epilepsies. This study aimed to analyze the genetic basis of a wide spectrum of epilepsies with age of onset spanning from the neonatal period to adulthood. A gene panel targeting 46 epilepsy genes was used on a cohort of 216 patients consecutively referred for panel testing. The patients had a range of different epilepsies from benign neonatal seizures to epileptic encephalopathies (EEs). Potentially causative variants were evaluated by literature and database searches, submitted to bioinformatic prediction algorithms, and validated by Sanger sequencing. If possible, parents were included for segregation analysis. We identified a presumed disease-causing variant in 49 (23%) of the 216 patients. The variants were found in 19 different genes including SCN1A, STXBP1, CDKL5, SCN2A, SCN8A, GABRA1, KCNA2, and STX1B. Patients with neonatal-onset epilepsies had the highest rate of positive findings (57%). The overall yield for patients with EEs was 32%, compared to 17% among patients with generalized epilepsies and 16% in patients with focal or multifocal epilepsies. By the use of a gene panel consisting of 46 epilepsy genes, we were able to find a disease-causing genetic variation in 23% of the analyzed patients. The highest yield was found among patients with neonatal-onset epilepsies and EEs.

Association between genes on chromosome 19p13.2 and panic disorder.

Panic disorder (PD) is a severe and disabling mental disorder, which is moderately heritable. In a previous study, we carried out a genome-wide association study using patients with PD and control individuals from the isolated population of the Faroe Islands and identified chromosome 19p13.2 as a candidate region. To further investigate this chromosomal region for association with PD, we analysed eight single nucleotide polymorphisms (SNPs) in three candidate genes - small-nuclear RNA activating complex, polypeptide 2 (SNAPC2), mitogen-activated protein kinase kinase 7 (MAP2K7) and leucine-rich repeat containing 8 family, member E (LRRC8E) - these genes have previously been directly or indirectly implicated in other mental disorders. A total of 511 patients with PD and 1029 healthy control individuals from the Faroe Islands, Denmark and Germany were included in the current study. SNPs covering the gene region of SNAPC2, MAP2K7 and LRRC8E were genotyped and tested for association with PD. In the Faroese cohort, rs7788 within SNAPC2 was significantly associated with PD, whereas rs3745383 within LRRC8E was nominally associated. No association was observed between the analysed SNPs and PD in the Danish cohorts. In the German women, we observed a nominal association between rs4804833 within MAP2K7 and PD. We present further evidence that chromosome 19p13.2 may harbour candidate genes that contribute towards the risk of developing PD. Moreover, the implication of the associated genes in other mental disorders may indicate shared genetic susceptibility between mental disorders. We show that associated variants may be sex specific, indicating the importance of carrying out a sex-specific association analysis of PD.

Phenotypic spectrum of GABRA1: From generalized epilepsies to severe epileptic encephalopathies.

OBJECTIVE: To delineate phenotypic heterogeneity, we describe the clinical features of a cohort of patients with GABRA1 gene mutations. METHODS: Patients with GABRA1 mutations were ascertained through an international collaboration. Clinical, EEG, and genetic data were collected. Functional analysis of 4 selected mutations was performed using the Xenopus laevis oocyte expression system. RESULTS: The study included 16 novel probands and 3 additional family members with a disease-causing mutation in the GABRA1 gene. The phenotypic spectrum varied from unspecified epilepsy (1), juvenile myoclonic epilepsy (2), photosensitive idiopathic generalized epilepsy (1), and generalized epilepsy with febrile seizures plus (1) to severe epileptic encephalopathies (11). In the epileptic encephalopathy group, the patients had seizures beginning between the first day of life and 15 months, with a mean of 7 months. Predominant seizure types in all patients were tonic-clonic in 9 participants (56%) and myoclonic seizures in 5 (31%). EEG showed a generalized photoparoxysmal response in 6 patients (37%). Four selected mutations studied functionally revealed a loss of function, without a clear genotype-phenotype correlation. CONCLUSIONS: GABRA1 mutations make a significant contribution to the genetic etiology of both benign and severe epilepsy syndromes. Myoclonic and tonic-clonic seizures with pathologic response to photic stimulation are common and shared features in both mild and severe phenotypes.

The contribution of next generation sequencing to epilepsy genetics.

During the last decade, next generation sequencing technologies such as targeted gene panels, whole exome sequencing and whole genome sequencing have led to an explosion of gene identifications in monogenic epilepsies including both familial epilepsies and severe epilepsies, often referred to as epileptic encephalopathies. The increased knowledge about causative genetic variants has had a major impact on diagnosis of genetic epilepsies and has already been translated into treatment recommendations for a few genes. This article provides an overview of how next generation sequencing has advanced our understanding of epilepsy genetics and discusses some of the recently discovered genes in monogenic epilepsies.

Primary functions of the quadratus femoris and obturator externus muscles indicated from lengths and moment arms measured in mobilized cadavers.

BACKGROUND: The small muscles of the pelvis and hip are often implicated in painful conditions. Although the quadratus femoris and obturator externus are usually described as external rotators of the hip, little is known about how they change their lengths and moment arms during human movement. Therefore, more precise measurements defining the positions and directions for their maximal strength and stretch are needed to better describe their functions and guide the clinical approach to pain. METHODS: Repeated measurements of the muscle lengths and range of motion were obtained using wires simulating dissected muscles on human cadaver hips. The lengths were measured at every 15° of flexion with and without maximal range of ab/adduction, rotation, and combinations of the two motions. Measurements were obtained from normal hips (n=3), and movement-lengthening relations were later differentiated into movement-moment arm relations. FINDINGS: The quadratus femoris showed maximum lengthening by flexion, adduction or abduction, and internal rotation, with the largest moment arms observed for extension in the deduced force-length efficient range of 60-90° flexion. The obturator externus showed maximum lengthening by extension, abduction, and internal rotation, with the largest moment arms observed for flexion and adduction in the deduced force-length efficient range around the hip's neutral position. INTERPRETATION: Our findings indicate that maximal strength of the quadratus femoris muscle will be delivered in a flexed position towards extension, while maximal strength of the obturator externus muscle will be delivered in an extended position towards flexion and adduction.

Clinical Phenotype of De Novo GNAO1 Mutation: Case Report and Review of Literature.

Mutations in the guanine nucleotide-binding protein (G protein), α activating activity polypeptide O (GNAO1) gene have recently been described in 6 patients with early infantile epileptic encephalopathies. In the present study, we report the phenotype and the clinical course of a 4-year-old female with an epileptic encephalopathy (Ohtahara syndrome) and profound intellectual disability due to a de novo GNAO1 mutation (c.692A>G; p.Tyr231Cys). Ohtahara syndrome is a devastating early infantile epileptic encephalopathy that can be caused by mutations in different genes, now also including GNAO1. The mutation was found using a targeted next generation sequencing gene panel and demonstrates targeted sequencing as a powerful tool for identifying mutations in genes where only a few de novo mutations have been identified.

Lengths of the external hip rotators in mobilized cadavers indicate the quadriceps coxa as a primary abductor and extensor of the flexed hip.

BACKGROUND: The primary function of the external rotators of the hip is inadequately described. The descriptions for peak strength and stretch take no account of how these muscles change their length during normal movement. The latter relationship is known to greatly influence contraction forces and reflect moment arms. The aim of the present study was to indicate positions and directions for peak strength and stretch of piriformis and obturator internus (including the gemelli), collectively defined as the quadriceps coxa, by measuring their changes in length due to normal movements. METHODS: Repeated measurements of muscle lengths and range of motions were acquired from dissected muscles on human cadaver hips. We measured at every 15° of flexion with and without adding end ab/adduction, rotations, and combinations thereof. Measurements were taken in three normal hips (1 female aged 59 years, 2 males aged 68 and 70 years) using a custom-engineered frame, electronic calipers, goniometer, and a string muscle model. Movement-lengthening relations were differentiated into movement-moment arm relations. FINDINGS: The piriformis and obturator internus were maximally lengthened (35 and 30mm) by 105° flexion and 10° adduction and relaxed by extension and abduction. With significant moment arms for extension and abduction in the movement-lengthening range deduced as force-efficient, our findings indicate peak strength by extension and abduction at 60° to 90° flexed positions. INTERPRETATION: This cadaver study indicates that the quadriceps coxa is a primary abductor and extensor from flexed positions, a strength function which may be of major importance in rising and propulsive motions.

Are TMEM genes potential candidate genes for panic disorder?

We analysed single nucleotide polymorphisms in two transmembrane genes (TMEM98 and TMEM132E) in panic disorder (PD) patients and control individuals from the Faroe Islands, Denmark and Germany. The genes encode single-pass membrane proteins and are located within chromosome 17q11.2-q12, a previously reported candidate region for PD. Three single nucleotide polymorphisms (rs887231, rs887230 and rs4795942) located upstream and within TMEM132E showed a nominal significant association with PD primarily in the Danish cohort. No nominal significant associations were observed between TMEM98 and PD. Our data indicate that TMEM132E might contribute moderately towards the risk of developing PD.

A genome-wide study of panic disorder suggests the amiloride-sensitive cation channel 1 as a candidate gene.

Panic disorder (PD) is a mental disorder with recurrent panic attacks that occur spontaneously and are not associated to any particular object or situation. There is no consensus on what causes PD. However, it is recognized that PD is influenced by environmental factors, as well as genetic factors. Despite a significant hereditary component, genetic studies have only been modestly successful in identifying genes of importance for the development of PD. In this study, we conducted a genome-wide scan using microsatellite markers and PD patients and control individuals from the isolated population of the Faroe Islands. Subsequently, we conducted a fine mapping, which revealed the amiloride-sensitive cation channel 1 (ACCN1) located on chromosome 17q11.2-q12 as a potential candidate gene for PD. The further analyses of the ACCN1 gene using single-nucleotide polymorphisms (SNPs) revealed significant association with PD in an extended Faroese case-control sample. However, analyses of a larger independent Danish case-control sample yielded no substantial significant association. This suggests that the possible risk alleles associated in the isolated population are not those involved in the development of PD in a larger outbred population.

Effect of training with different mechanical loadings on MyHC and GLUT4 changes.

PURPOSE: There is an inverse relationship between insulin sensitivity and percentage of myosin heavy chain IIx (MyHC IIx) isoform in sedentary, obese, and type 2 diabetic humans. How different exercise conditions may reduce the proportion of MyHC IIx and in parallel elevate glucose transporter 4 (GLUT4) content is interesting in a therapeutic setting. This study investigates the nature of exercise signals regulating MyHC gene switching and whether it is accompanied by GLUT4 changes. METHODS: Thirty-two subjects performed high loading (60% of 1 repetition maximum [RM]) or low loading (30% of 1 RM) elbow extensions in a training apparatus and exercised three times per week for either 5 wk (low volume) or 8 wk (high volume). MyHC and GLUT4 contents in the musculus triceps brachii were measured by Western blotting pre- and posttraining and after 8 wk of detraining. RESULTS: All training regimes resulted in MyHC IIx changes of similar magnitude, and differences in training volume had no effect on the outcome. The reduction in MyHC IIx content after high loading, high volume was similar to low loading, matching volume of training. Thus, there was no effect of training load on MyHC changes. GLUT4 increased more after high than low loading (P < 0.0.1). In addition, the larger increases in the GLUT4 were associated with the larger reductions in MyHC IIx content (r = -0.56, P < 0.01). Detraining returned GLUT4 to baseline, but MyHC IIx content was still higher than baseline (P < 0.01). CONCLUSION: Magnitude of loading is not important for suppression of MyHC IIx but for increases in GLUT4 content. The GLUT4 content responded, however, more rapidly to detraining than the MyHC IIx and IIa isoforms.

Effect of training with different intensities and volumes on muscle fibre enzyme activity and cross sectional area in the m. triceps brachii.

This study primarily examined how intermittent versus continuous endurance training, using similar or dissimilar volumes, affected muscle fibre enzyme activities in the triceps brachii muscle. Thirty-two subjects performed either intermittent (60% of 1RM) or continuous (30% of 1RM) elbow extensions 3 times week(-1) in a training apparatus. Training was performed until either a low (five) or a high volume (8 weeks) was accumulated. Muscle biopsies from the m. triceps brachii were taken pre- and post training and following 8 weeks of detraining. Marker enzymes for muscle fibre oxidative (succinate dehydrogenase SDH) and glycolytic (glycerophosphate dehydrogenase; alpha-GPDH) capacity was assessed by histochemistry, and the resulting enzyme activities measured by image analysis. The type of training affected enzyme activities differently. In type 1 fibres, continuous and intermittent training was equally effective in increasing SDH activity, while intermittent training increased SDH activity more than continuous training in type 2 fibres (P < 0.05). Intermittent training increased alpha-GPDH activity more than continuous training both in type 1 (P < 0.001) and type 2 fibres (P < 0.05), but the increase in glycolytic capacity following intermittent training was larger in type 1 (54%) than in type 2 fibres (23%). There was no effect of training volume on oxidative or glycolytic capacity in either fibre type. Thus, when training intensity is sufficient to stimulate to increases in oxidative and glycolytic capacity, the SDH and alpha-GPDH response seems to be volume independent. Detraining reduced Post-T enzyme activities to baseline (all; P < 0.01).

Effect of training and detraining on the expression of heat shock proteins in m. triceps brachii of untrained males and females.

Forty untrained persons were randomized to four different training protocols that exercised the m. triceps brachii. Group 1 and 2 performed high intensity (HI) elbow extensions and group 3 and 4 performed low intensity (LI) elbow extensions. Group 1 and 3 trained until they had accumulated a matching high volume (HV) of training, while group 2 and 4 trained until they had accumulated a matching low volume (LV) of training. Training for 5-8 weeks increased the HSP72, HSP27 and GRP75 levels in the subjects' m. triceps brachii by 111, 71 and 192%, respectively (Fig. 1a-c). There were, however, no significant differences in the heat shock protein (HSP) responses to training between the four training groups (Fig. 2a-c). The frequency of extreme responses to exercise was, however, higher after HI exercise than after LI exercise, indicating that HI exercise induces extreme HSP reactions in some subjects. When we assigned the subjects to three clusters, according to the total number of repetitions they had lifted, the subjects who had lifted the highest number of repetitions had lower PostExc HSP levels compared with subjects that lifted the lowest number of repetitions (Fig. 3a-c). Additionally, there was a negative non-linear regression (Fig. 4a-c) between the subjects PreExc levels of HSP72, HSP27 and GRP75 and the percentage change in their respective protein concentration after training (r = -0.75, -0.89 and -0.88, all P < 0.0001). Thus, the PreExc level of HSPs seems to be an important "regulator" of HSP expression following the training.

Effect of concentric or eccentric weight training on the expression of heat shock proteins in m. biceps brachii of very well trained males.

Increased HSP expression in response to acute exercise is well documented in animal studies, and there is growing evidence that similar responses occur in man. In general, many human exercise studies have investigated the HSP response to low force continuous activity, while the knowledge about the HSP response to high force intermittent type of activity, like weight training, is so far sparse. In addition, most studies have used untrained subjects, and a common observation is that acute low force continuous activity in untrained individuals increases the HSP expression in these individuals. The main scope of this study was to investigate the HSP response in very well trained males subjected to longitudinal high intensity exercise, and if this response was dependent on exercise modality [i.e. eccentric (ECC) or concentric (CON) contractions]. Very well trained males performed progressive strength training consisting of either high force ECC or high force CON elbow flexions 2-3 times a week for 12 weeks. Compared with pre-exercise levels, HSP72 expression decreased by 46.1% (P<0.05) after CON contractions. GRP75 expression was unchanged after ECC or CON contractions, while ubiquitin expression decreased by 19.9% (P<0.02) after ECC contractions. These findings imply that chronic, intensive exercise may attenuate the HSP response in well-trained males.