Seo-Kyung Chung

Pathophysiological Mechanisms of Dominant and Recessive GLRA1 Mutations in Hyperekplexia

Hyperekplexia is a rare, but potentially fatal, neuromotor disorder characterized by exaggerated startle reflexes and hypertonia in response to sudden, unexpected auditory or tactile stimuli. This disorder is primarily caused by inherited mutations in the genes encoding the glycine receptor (GlyR) α1 subunit (GLRA1) and the presynaptic glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of GLRA1 in 88 new unrelated human hyperekplexia patients revealed 19 sequence variants in 30 index cases, of which 21 cases were inherited in recessive or compound heterozygote modes. This indicates that recessive hyperekplexia is far more prevalent than previous estimates. From the 19 GLRA1 sequence variants, we have investigated the functional effects of 11 novel and 2 recurrent mutations. The expression levels and functional properties of these hyperekplexia mutants were analyzed using a high-content imaging system and patch-clamp electrophysiology. When expressed in HEK293 cells, either as homomeric α1 or heteromeric α1β GlyRs, subcellular localization defects were the major mechanism underlying recessive mutations. However, mutants without trafficking defects typically showed alterations in the glycine sensitivity suggestive of disrupted receptor function. This study also reports the first hyperekplexia mutation associated with a GlyR leak conductance, suggesting tonic channel opening as a new mechanism in neuronal ligand-gated ion channels.

Overlapping cortical malformations and mutations in TUBB2B and TUBA1A

Polymicrogyria and lissencephaly are causally heterogeneous disorders of cortical brain development, with distinct neuropathological and neuroimaging patterns. They can be associated with additional structural cerebral anomalies, and recurrent phenotypic patterns have led to identification of recognizable syndromes. The lissencephalies are usually single-gene disorders affecting neuronal migration during cerebral cortical development. Polymicrogyria has been associated with genetic and environmental causes and is considered a malformation secondary to abnormal post-migrational development. However, the aetiology in many individuals with these cortical malformations is still unknown. During the past few years, mutations in a number of neuron-specific α- and β-tubulin genes have been identified in both lissencephaly and polymicrogyria, usually associated with additional cerebral anomalies including callosal hypoplasia or agenesis, abnormal basal ganglia and cerebellar hypoplasia. The tubulin proteins form heterodimers that incorporate into microtubules, cytoskeletal structures essential for cell motility and function. In this study, we sequenced the TUBB2B and TUBA1A coding regions in 47 patients with a diagnosis of polymicrogyria and five with an atypical lissencephaly on neuroimaging. We identified four β-tubulin and two α-tubulin mutations in patients with a spectrum of cortical and extra-cortical anomalies. Dysmorphic basal ganglia with an abnormal internal capsule were the most consistent feature. One of the patients with a TUBB2B mutation had a lissencephalic phenotype, similar to that previously associated with a TUBA1A mutation. The remainder had a polymicrogyria-like cortical dysplasia, but the grey matter malformation was not typical of that seen in classical polymicrogyria. We propose that the cortical malformations associated with these genes represent a recognizable tubulinopathy-associated spectrum that ranges from lissencephalic to polymicrogyric cortical dysplasias, suggesting shared pathogenic mechanisms in terms of microtubular function and interaction with microtubule-associated proteins.

Identification of large gene deletions and duplications in KCNQ1 and KCNH2 in patients with long QT syndrome

BACKGROUNDnSequencing or denaturing high-performance liquid chromatography (dHPLC) analysis of the known genes associated with the long QT syndrome (LQTS) fails to identify mutations in approximately 25% of subjects with inherited LQTS. Large gene deletions and duplications can be missed with these methodologies.nnnOBJECTIVEnThe purpose of this study was to determine whether deletions and/or duplications of one or more exons of the main LQTS genes were present in an LQTS mutation-negative cohort.nnnMETHODSnMultiplex ligation-dependent probe amplification (MLPA), a quantitative fluorescent approach, was used to screen 26 mutation-negative probands with an unequivocal LQTS phenotype (Schwartz score >4). The appropriate MLPA kit contained probes for selected exons in LQTS genes KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2. Real-time polymerase chain reaction was used to validate the MLPA findings.nnnRESULTSnAltered exon copy number was detected in 3 (11.5%) patients: (1) an ex13-14del of the KCNQ1 gene in an 11-year-old boy with exercise-induced collapse (QTc 580 ms); (2) an ex6-14del of the KCNH2 gene in a 22-year-old woman misdiagnosed with epilepsy since age 9 years (QTc 560 ms) and a sibling with sudden death at age 13 years; and (3) an ex9-14dup of the KCNH2 gene in a 12 year-old boy (QTc 550 ms) following sudden nocturnal death of his 32-year-old mother.nnnCONCLUSIONnIf replicated, this study demonstrates that more than 10% of patients with LQTS and a negative current generation genetic test have large gene deletions or duplications among the major known LQTS susceptibility genes. As such, these findings suggest that sequencing-based mutation detection strategies should be followed by deletion/duplication screening in all LQTS mutation-negative patients.

De Novo Mutations in SLC1A2 and CACNA1A Are Important Causes of Epileptic Encephalopathies

Epileptic encephalopathies (EEs) are the most clinically important group of severe early-onset epilepsies. Next-generation sequencing has highlighted the crucial contribution of de novo mutations to the genetic architecture of EEs as well as to their underlying genetic heterogeneity. Our previous whole-exome sequencing study of 264 parent-child trios revealed more than 290 candidate genes in which only a single individual had a de novo variant. We sought to identify additional pathogenic variants in a subset (n = 27) of these genes via targeted sequencing in an unsolved cohort of 531 individuals with a diverse range of EEs. We report 17 individuals with pathogenic variants in seven of the 27 genes, defining a genetic etiology in 3.2% of this unsolved cohort. Our results provide definitive evidence that de novo mutations in SLC1A2 and CACNA1A cause specific EEs and expand the compendium of clinically relevant genotypes for GABRB3. We also identified EEs caused by genetic variants in ALG13, DNM1, and GNAO1 and report a mutation in IQSEC2. Notably, recurrent mutations accounted for 7/17 of the pathogenic variants identified. As a result of high-depth coverage, parental mosaicism was identified in two out of 14 cases tested with mutant allelic fractions of 5%-6% in the unaffected parents, carrying significant reproductive counseling implications. These results confirm that dysregulation in diverse cellular neuronal pathways causes EEs, and they will inform the diagnosis and management of individuals with these devastating disorders.

Ultra-rare genetic variation in common epilepsies: a case-control sequencing study

BACKGROUNDnDespite progress in understanding the genetics of rare epilepsies, the more common epilepsies have proven less amenable to traditional gene-discovery analyses. We aimed to assess the contribution of ultra-rare genetic variation to common epilepsies.nnnMETHODSnWe did a case-control sequencing study with exome sequence data from unrelated individuals clinically evaluated for one of the two most common epilepsy syndromes: familial genetic generalised epilepsy, or familial or sporadic non-acquired focal epilepsy. Individuals of any age were recruited between Nov 26, 2007, and Aug 2, 2013, through the multicentre Epilepsy Phenome/Genome Project and Epi4K collaborations, and samples were sequenced at the Institute for Genomic Medicine (New York, USA) between Feb 6, 2013, and Aug 18, 2015. To identify epilepsy risk signals, we tested all protein-coding genes for an excess of ultra-rare genetic variation among the cases, compared with control samples with no known epilepsy or epilepsy comorbidity sequenced through unrelated studies.nnnFINDINGSnWe separately compared the sequence data from 640 individuals with familial genetic generalised epilepsy and 525 individuals with familial non-acquired focal epilepsy to the same group of 3877 controls, and found significantly higher rates of ultra-rare deleterious variation in genes established as causative for dominant epilepsy disorders (familial genetic generalised epilepsy: odd ratio [OR] 2·3, 95% CI 1·7-3·2, p=9·1u2008×u200810-8; familial non-acquired focal epilepsy 3·6, 2·7-4·9, p=1·1u2008×u200810-17). Comparison of an additional cohort of 662 individuals with sporadic non-acquired focal epilepsy to controls did not identify study-wide significant signals. For the individuals with familial non-acquired focal epilepsy, we found that five known epilepsy genes ranked as the top five genes enriched for ultra-rare deleterious variation. After accounting for the control carrier rate, we estimate that these five genes contribute to the risk of epilepsy in approximately 8% of individuals with familial non-acquired focal epilepsy. Our analyses showed that no individual gene was significantly associated with familial genetic generalised epilepsy; however, known epilepsy genes had lower p values relative to the rest of the protein-coding genes (p=5·8u2008×u200810-8) that were lower than expected from a random sampling of genes.nnnINTERPRETATIONnWe identified excess ultra-rare variation in known epilepsy genes, which establishes a clear connection between the genetics of common and rare, severe epilepsies, and shows that the variants responsible for epilepsy risk are exceptionally rare in the general population. Our results suggest that the emerging paradigm of targeting of treatments to the genetic cause in rare devastating epilepsies might also extend to a proportion of common epilepsies. These findings might allow clinicians to broadly explain the cause of these syndromes to patients, and lay the foundation for possible precision treatments in the future.nnnFUNDINGnNational Institute of Neurological Disorders and Stroke (NINDS), and Epilepsy Research UK.

De Novo Mutations in the Beta-Tubulin Gene TUBB2A Cause Simplified Gyral Patterning and Infantile-Onset Epilepsy

Tubulins, and microtubule polymers into which they incorporate, play critical mechanical roles in neuronal function during cell proliferation, neuronal migration, and postmigrational development: the three major overlapping events of mammalian cerebral cortex development. A number of neuronally expressed tubulin genes are associated with a spectrum of disorders affecting cerebral cortex formation. Such tubulinopathies include lissencephaly/pachygyria, polymicrogyria-like malformations, and simplified gyral patterns, in addition to characteristic extracortical features, such as corpus callosal, basal ganglia, and cerebellar abnormalities. Epilepsy is a common finding in these related disorders. Here we describe two unrelated individuals with infantile-onset epilepsy and abnormalities of brain morphology, harboring de novo variants that affect adjacent amino acids in a beta-tubulin gene TUBB2A. Located in a highly conserved loop, we demonstrate impaired tubulin and microtubule function resulting from each variant in vitro and by using in silico predictive modeling. We propose that the affected functional loop directly associates with the alpha-tubulin-bound guanosine triphosphate (GTP) molecule, impairing the intradimer interface and correct formation of the alpha/beta-tubulin heterodimer. This study associates mutations in TUBB2A with the spectrum of tubulinopathy phenotypes. As a consequence, genetic variations affecting all beta-tubulin genes expressed at high levels in the brain (TUBB2B, TUBB3, TUBB, TUBB4A, and TUBB2A) have been linked with malformations of cortical development.

Biophysical Properties of 9 KCNQ1 Mutations Associated With Long-QT Syndrome

Background—Inherited long-QT syndrome is characterized by prolonged QT interval on the ECG, syncope, and sudden death caused by ventricular arrhythmia. Causative mutations occur mostly in cardiac potassium and sodium channel subunit genes. Confidence in mutation pathogenicity is usually reached through family genotype-phenotype tracking, control population studies, molecular modeling, and phylogenetic alignments; however, biophysical testing offers a higher degree of validating evidence. Methods and Results—By using in vitro electrophysiological testing of transfected mutant and wild-type long-QT syndrome constructs into Chinese hamster ovary cells, we investigated the biophysical properties of 9 KCNQ1 missense mutations (A46T, T265I, F269S, A302V, G316E, F339S, R360G, H455Y, and S546L) identified in a New Zealand–based long-QT syndrome screening program. We demonstrate through electrophysiology and molecular modeling that 7 of the missense mutations have profound pathological dominant-negative loss-of-function properties, confirming their likely disease-causing nature. This supports the use of these mutations in diagnostic family screening. Two mutations (A46T, T265I) show suggestive evidence of pathogenicity within the experimental limits of biophysical testing, indicating that these variants are disease-causing via delayed- or fast-activation kinetics. Further investigation of the A46T family has revealed an inconsistent cosegregation of the variant with the clinical phenotype. Conclusions—Electrophysiological characterization should be used to validate long-QT syndrome pathogenicity of novel missense channelopathies. When such results are inconclusive, great care should be taken with genetic counseling and screening of such families, and alternative disease-causing mechanisms should be considered.

A novel GABRG2 mutation, p.R136*, in a family with GEFS+ and extended phenotypes.

Genetic mutations in voltage-gated and ligand-gated ion channel genes have been identified in a small number of Mendelian families with genetic generalised epilepsies (GGEs). They are commonly associated with febrile seizures (FS), childhood absence epilepsy (CAE) and particularly with generalised or genetic epilepsy with febrile seizures plus (GEFS+). In clinical practice, despite efforts to categorise epilepsy and epilepsy families into syndromic diagnoses, many generalised epilepsies remain unclassified with a presumed genetic basis. During the systematic collection of epilepsy families, we assembled a cohort of families with evidence of GEFS+ and screened for variations in the γ2 subunit of the γ-aminobutyric acid (GABA) type A receptor gene (GABRG2). We detected a novel GABRG2(p.R136*) premature translation termination codon in one index-case from a two-generation nuclear family, presenting with an unclassified GGE, a borderline GEFS+ phenotype with learning difficulties and extended behavioural presentation. The GABRG2(p.R136*) mutation segregates with the febrile seizure component of this familys GGE and is absent in 190 healthy control samples. In vitro expression assays demonstrated that γ2(p.R136*) subunits were produced, but had reduced cell-surface and total expression. When γ2(p.R136*) subunits were co-expressed with α1 and β2 subunits in HEK 293T cells, GABA-evoked currents were reduced. Furthermore, γ2(p.R136*) subunits were highly-expressed in intracellular aggregations surrounding the nucleus and endoplasmic reticulum (ER), suggesting compromised receptor trafficking. A novel GABRG2(p.R136*) mutation extends the spectrum of GABRG2 mutations identified in GEFS+ and GGE phenotypes, causes GABAA receptor dysfunction, and represents a putative epilepsy mechanism.

Elevated serum gastrin levels in Jervell and Lange-Nielsen syndrome: A marker of severe KCNQ1 dysfunction?

BACKGROUNDnThe potassium channel I(Ks), which is encoded by the KCNQ1 gene, is expressed in organ systems including the inner ear, kidneys, lungs, intestine, and stomach in addition to the heart. Increasing evidence indicates that I(Ks) in the stomach plays an essential role in enabling gastric acid production. It is not known whether gastric acid production is disordered in patients with long QT type 1. Serum gastrin levels become elevated in subjects with disordered gastric acid production.nnnOBJECTIVEnThe purpose of this study was to evaluate serum gastrin levels, as a surrogate for impaired gastric acid secretion, in patients with KCNQ1 mutations, and to see if gastrin levels correlate with severity of cardiac disease.nnnMETHODSnFasting serum gastrin levels were measured using a standardized immunometric technique in an index case and 12 subjects with known KCNQ1 mutations.nnnRESULTSnAn adult female with Jervell and Lange-Nielsen syndrome (JLNS; with KCNQ1 nonsense mutations p.Arg518X and p.Arg190AlafsX95 ) presented with multiple gastric carcinoid tumors and grossly elevated serum gastrin levels (943-1,570 pmol/L; normal 6-55 pmol/L) and absent acid secretion. Gastrin levels in two girls with JLNS, unrelated to the index case (missense mutations p.Leu266Pro and Gly269Ser), also were high (305 and 229 pmol/L). Gastrin levels were normal in 10 KCNQ1 heterozygous single mutation carriers, even in those with severe long QT syndrome, including three heterozygous family members of the JLNS subjects.nnnCONCLUSIONnJLNS may be associated with elevated gastrin levels, impaired acid secretion, and risk of gastric carcinoid tumors. Among KCNQ1 single mutation carriers, gastrin levels were normal and did not appear to be linked to the severity of clinical expression of long QT syndrome.

Pathogenic copy number variants and SCN1A mutations in patients with intellectual disability and childhood-onset epilepsy.

BackgroundCopy number variants (CNVs) have been linked to neurodevelopmental disorders such as intellectual disability (ID), autism, epilepsy and psychiatric disease. There are few studies of CNVs in patients with both ID and epilepsy.MethodsWe evaluated the range of rare CNVs found in 80 Welsh patients with ID or developmental delay (DD), and childhood-onset epilepsy. We performed molecular cytogenetic testing by single nucleotide polymorphism array or microarray-based comparative genome hybridisation.Results8.8xa0% (7/80) of the patients had at least one rare CNVs that was considered to be pathogenic or likely pathogenic. The CNVs involved known disease genes (EHMT1, MBD5 and SCN1A) and imbalances in genomic regions associated with neurodevelopmental disorders (16p11.2, 16p13.11 and 2q13). Prompted by the observation of two deletions disrupting SCN1A we undertook further testing of this gene in selected patients. This led to the identification of four pathogenic SCN1A mutations in our cohort.ConclusionsWe identified five rare de novo deletions and confirmed the clinical utility of array analysis in patients with ID/DD and childhood-onset epilepsy. This report adds to our clinical understanding of these rare genomic disorders and highlights SCN1A mutations as a cause of ID and epilepsy, which can easily be overlooked in adults.