K.L.I. van Gassen

High-resolution genetic mapping of mammalian motor activity levels in mice

The generation of motor activity levels is under tight neural control to execute essential behaviors, such as movement toward food or for social interaction. To identify novel neurobiological mechanisms underlying motor activity levels, we studied a panel of chromosome substitution (CS) strains derived from mice with high (C57BL/6J strain) or low motor activity levels (A/J strain) using automated home cage behavioral registration. In this study, we genetically mapped the expression of baseline motor activity levels (horizontal distance moved) to mouse chromosome 1. Further genetic mapping of this trait revealed an 8.3‐Mb quantitative trait locus (QTL) interval. This locus is distinct from the QTL interval for open‐field anxiety‐related motor behavior on this chromosome. By data mining, an existing phenotypic and genotypic data set of 2445 genetically heterogeneous mice (http://gscan.well.ox.ac.uk/), we confirmed linkage to the peak marker at 79 970 253 bp and refined the QTL to a 312‐kb interval containing a single gene (A830043J08Rik). Sequence analysis showed a nucleotide deletion in the 3′ untranslated region of the Riken gene. Genome‐wide microarray gene expression profiling in brains of discordant F2 individuals from CS strain 1 showed a significant upregulation of Epha4 in low‐active F2 individuals. Inclusion of a genetic marker for Epha4 confirmed that this gene is located outside of the QTL interval. Both Epha4 and A830043J08Rik are expressed in brain motor circuits, and similar to Epha4 mutants, we found linkage between reduced motor neurons number and A/J chromosome 1. Our findings provide a novel QTL and a potential downstream target underlying motor circuitry development and the expression of physical activity levels.

Characterization of febrile seizures and febrile seizure susceptibility in mouse inbred strains

Febrile seizures (FS) are the most prevalent seizures in children. Although FS are largely benign, complex FS increase the risk to develop temporal lobe epilepsy (TLE). Studies in rat models for FS have provided information about functional changes in the hippocampus after complex FS. However, our knowledge about the genes and pathways involved in the causes and consequences of FS is still limited. To enable molecular, genetic and knockout studies, we developed and characterized an FS model in mice and used it as a phenotypic screen to analyze FS susceptibility. Hyperthermia was induced by warm air in 10‐ to 14‐day‐old mice and induced FS in all animals. Under the conditions used, seizure‐induced behavior in mice and rats was similar. In adulthood, treated mice showed increased hippocampal Ih current and seizure susceptibility, characteristics also seen after FS in rats. Of the seven genetically diverse mouse strains screened for FS susceptibility, C57BL/6J mice were among the most susceptible, whereas A/J mice were among the most resistant. Strains genetically similar to C57BL/6J also showed a susceptible phenotype. Our phenotypic data suggest that complex genetics underlie FS susceptibility and show that the C57BL/6J strain is highly susceptible to FS. As this strain has been described as resistant to convulsants, our data indicate that susceptibility genes for FS and convulsants are distinct. Insight into the mechanisms underlying seizure susceptibility and FS may help to identify markers for the early diagnosis of children at risk for complex FS and TLE and may provide new leads for treatment.

The chemokine CCL2 modulates Ca2+ dynamics and electrophysiological properties of cultured cerebellar Purkinje neurons.

The chemokine CCL2 is produced at high levels in the central nervous system (CNS) during infection, injury, neuroinflammation and other pathological conditions. Cells of the CNS including neurons and glia express receptors for CCL2 and these receptors may contribute to a signaling system through which pathologic conditions in the CNS are communicated. However, our understanding of the consequences of activation of chemokine signaling in the CNS is limited, especially for neurons. In many cell types, chemokine signaling alters intracellular Ca2+ dynamics. Therefore, we investigated the potential involvement of this mechanism in neuronal signaling activated by CCL2. In addition, we examined the effects of CCL2 on neuronal excitability. The studies focused on the rat cerebellar Purkinje neuron, an identified CNS neuronal type reported to express both CCL2 and its receptor, CCR2. Immunohistochemical studies of Purkinje neurons in situ confirmed that they express CCR2 and CCL2. The effect of exogenous application on Purkinje neurons was studied in a cerebellar culture preparation. CCL2 was tested by micropressure or bath application, at high concentrations (13–100 nm) to simulate conditions during a pathologic state. Results show that Purkinje neurons express receptors for CCL2 and that activation of these receptors alters several neuronal properties. CCL2 increased resting Ca2+ levels, enhanced the Ca2+ response evoked by activation of metabotropic glutamate receptor 1 and depressed action potential generation in the cultured Purkinje neurons. Passive membrane properties were unaltered. These modulatory effects of CCL2 on neuronal properties are likely to contribute to the altered CNS function associated with CNS disease and injury.

Chronic exposure to the chemokine CCL3 enhances neuronal network activity in rat hippocampal cultures

We examined the effect of chronic CCL3 treatment on the properties of cultured rat hippocampal neurons to gain an understanding of the neuronal effects of CCL3 during neuroinflammatory disorders. Western blot assays showed that chronic exposure to CCL3 altered the level of specific neuronal and glial proteins and that CCL3 had no effect on neuronal survival. CCL3 treatment also altered intracellular Ca(2+) dynamics and increased Ca(2+) levels in hippocampal neurons, measured by fura-2 imaging techniques. Additionally, chronic CCL3 increased NMDA-evoked Ca(2+) signals in the hippocampal neurons and increased NMDA receptor levels. These CCL3-induced neuroadaptive changes could play an important role in the CNS dysfunction associated with CNS disorders with a neuroinflammatory component.

Haploinsufficiency of glutamine synthetase increases susceptibility to experimental febrile seizures.

Glutamine synthetase (GS) is a pivotal glial enzyme in the glutamate–glutamine cycle. GS is important in maintaining low extracellular glutamate concentrations and is downregulated in the hippocampus of temporal lobe epilepsy patients with mesial–temporal sclerosis, an epilepsy syndrome that is frequently associated with early life febrile seizures (FS). Human congenital loss of GS activity has been shown to result in brain malformations, seizures and death within days after birth. Recently, we showed that GS knockout mice die during embryonic development and that haploinsufficient GS mice have no obvious abnormalities or behavioral seizures. In the present study, we investigated whether reduced expression/activity of GS in haploinsufficient GS mice increased the susceptibility to experimentally induced FS. FS were elicited by warm‐air‐induced hyperthermia in 14‐day‐old mice and resulted in seizures in most animals. FS susceptibility was measured as latencies to four behavioral FS characteristics. Our phenotypic data show that haploinsufficient mice are more susceptible to experimentally induced FS (P < 0.005) than littermate controls. Haploinsufficient animals did not differ from controls in hippocampal amino acid content, structure (Nissl and calbindin), glial properties ( glial fibrillary acidic protein and vimentin) or expression of other components of the glutamate–glutamine cycle (excitatory amino acid transporter‐2 and vesicular glutamate transporter‐1). Thus, we identified GS as a FS susceptibility gene. GS activity‐disrupting mutations have been described in the human population, but heterozygote mutations were not clearly associated with seizures or epilepsy. Our results indicate that individuals with reduced GS activity may have reduced FS seizure thresholds. Genetic association studies will be required to test this hypothesis.

Phenotyping mouse chromosome substitution strains reveal multiple QTLs for febrile seizure susceptibility

Febrile seizures (FS) are the most common seizure type in children and recurrent FS are a risk factor for developing temporal lobe epilepsy. Although the mechanisms underlying FS are largely unknown, recent family, twin and animal studies indicate that genetics are important in FS susceptibility. Here, a forward genetic strategy was used employing mouse chromosome substitution strains (CSS) to identify novel FS susceptibility quantitative trait loci (QTLs). FS were induced by exposure to warm air at postnatal day 14. Video electroencephalogram monitoring identified tonic–clonic convulsion onset, defined as febrile seizure latency (FSL), as a reliable phenotypic parameter to determine FS susceptibility. FSL was determined in both sexes of the host strain (C57BL/6J), the donor strain (A/J) and CSS. C57BL/6J mice were more susceptible to FS than A/J mice. Phenotypic screening of the CSS panel identified six strains (CSS1, ‐2, ‐6 ‐10, ‐13 and ‐X) carrying QTLs for FS susceptibility. CSS1, ‐10 and ‐13 were less susceptible (protective QTLs), whereas CSS2, ‐6 and ‐X were more susceptible (susceptibility QTLs) to FS than the C57BL/6J strain. Our data show that mouse FS susceptibility is determined by complex genetics, which is distinct from that for chemically induced seizures. This is the first data set using CSS to screen for a seizure trait in mouse pups. It provides evidence for common FS susceptibility QTLs that serve as starting points to fine map FS susceptibility QTLs and to identify FS susceptibility genes. This will increase our understanding of human FS, working toward the identification of new therapeutic targets.

Points to consider for laboratories reporting results from diagnostic genomic sequencing

Although NGS technologies are well-embedded in the clinical setting for identification of genetic causes of disease, guidelines issued by professional bodies are inconsistent regarding some aspects of reporting results. Most recommendations do not give detailed guidance about whether variants of uncertain significance (VUS) should be reported by laboratory personnel to clinicians, and give conflicting messages regarding whether unsolicited findings (UF) should be reported. There are also differences both in their recommendations regarding whether actively searching for secondary findings (SF) is appropriate, and in the extent to which they address the duty (or lack thereof) to reanalyse variants when new information arises. An interdisciplinary working group considered the current guidelines, their own experiences, and data from a recent qualitative study to develop a set of points to consider for laboratories reporting results from diagnostic NGS. These points to consider fall under six categories: (i) Testing approaches and technologies used, (ii) Approaches for VUS; (iii) Approaches for reporting UF, (iv) Approaches regarding SF; (v) Reanalysis of data & re-contact; and vi) Minors. While it is unclear whether uniformity in reporting across all laboratories is desirable, we hope these points to consider will be useful to diagnostic laboratories as they develop their processes for making decisions about reporting VUS and UF from NGS in the diagnostic context.

Identification of C12orf4 as a gene for autosomal recessive intellectual disability

Intellectual disability (ID) is a major health problem in our society. Genetic causes of ID remain unknown because of its vast heterogeneity. Here we report two Finnish families and one Dutch family with affected individuals presenting with mild to moderate ID, neuropsychiatric symptoms and delayed speech development. By utilizing whole exome sequencing (WES), we identified a founder missense variant c.983T>C (p.Leu328Pro) in seven affected individuals from two Finnish consanguineous families and a deletion c.799_1034‐429delinsTTATGA (p.Gln267fs) in one affected individual from a consanguineous Dutch family in the C12orf4 gene on chromosome 12. Both the variants co‐segregated in the respective families as an autosomal recessive trait. Screening of the p.Leu328Pro variant showed enrichment in the North Eastern sub‐isolate of Finland among anonymous local blood donors with a carrier frequency of 1:53, similar to other disease mutations with a founder effect in that region. To date, only one Arab family with a three affected individuals with a frameshift insertion variant in C12orf4 has been reported. In summary, we expand and establish the clinical and mutational spectrum of C12orf4 variants. Our findings implicate C12orf4 as a causative gene for autosomal recessive ID.

De novo truncating variants in the intronless IRF2BPL are responsible for developmental epileptic encephalopathy

PurposeDevelopmental and epileptic encephalopathies (DEEs) are severe clinical conditions characterized by stagnation or decline of cognitive and behavioral abilities preceded, accompanied or followed by seizures. Because DEEs are clinically and genetically heterogeneous, next-generation sequencing, especially exome sequencing (ES), is becoming a first-tier strategy to identify the molecular etiologies of these disorders.MethodsWe combined ES analysis and international data sharing.ResultsWe identified 11 unrelated individuals with DEE and de novo heterozygous truncating variants in the interferon regulatory factor 2–binding protein-like gene (IRF2BPL). The 11 individuals allowed for delineation of a consistent neurodevelopmental disorder characterized by mostly normal initial psychomotor development followed by severe global neurological regression and epilepsy with nonspecific electroencephalogram (EEG) abnormalities and variable central nervous system (CNS) anomalies. IRF2BPL, also known as enhanced at puberty protein 1 (EAP1), encodes a transcriptional regulator containing a C-terminal RING-finger domain common to E3 ubiquitin ligases. This domain is required for its repressive and transactivating transcriptional properties. The variants identified are expected to encode a protein lacking the C-terminal RING-finger domain.ConclusionsThese data support the causative role of truncating IRF2BPL variants in pediatric neurodegeneration and expand the spectrum of transcriptional regulators identified as molecular factors implicated in genetic developmental and epileptic encephalopathies.

De novo variants in CDK13 associated with syndromic ID/DD: Molecular and clinical delineation of 15 individuals and a further review

De novo variants in the gene encoding cyclin‐dependent kinase 13 (CDK13) have been associated with congenital heart defects and intellectual disability (ID). Here, we present the clinical assessment of 15 individuals and report novel de novo missense variants within the kinase domain of CDK13. Furthermore, we describe 2 nonsense variants and a recurrent frame‐shift variant. We demonstrate the synthesis of 2 aberrant CDK13 transcripts in lymphoblastoid cells from an individual with a splice‐site variant. Clinical characteristics of the individuals include mild to severe ID, developmental delay, behavioral problems, (neonatal) hypotonia and a variety of facial dysmorphism. Congenital heart defects were present in 2 individuals of the current cohort, but in at least 42% of all known individuals. An overview of all published cases is provided and does not demonstrate an obvious genotype‐phenotype correlation, although 2 individuals harboring a stop codons at the end of the kinase domain might have a milder phenotype. Overall, there seems not to be a clinically recognizable facial appearance. The variability in the phenotypes impedes an à vue diagnosis of this syndrome and therefore genome‐wide or gene‐panel driven genetic testing is needed. Based on this overview, we provide suggestions for clinical work‐up and management of this recently described ID syndrome.