KCNT2-Related Disorders: Phenotypes, Functional, and Pharmacological Properties.

OBJECTIVE: Pathogenic variants in KCNT2 are rare causes of developmental epileptic encephalopathy (DEE). We herein describe the phenotypic and genetic features of patients with KCNT2-related DEE, and the in vitro functional and pharmacological properties of KCNT2 channels carrying 14 novel or previously untested variants. METHODS: Twenty-five patients harboring KCNT2 variants were investigated: 12 were identified through an international collaborative network, 13 were retrieved from the literature. Clinical data were collected and included in a standardized phenotyping sheet. Novel variants were detected using exome sequencing and classified using ACMG criteria. Functional and pharmacological studies were performed by whole-cell electrophysiology in HEK-293 and SH-SY5Y cells. RESULTS: The phenotypic spectrum encompassed: (a) intellectual disability/developmental delay (21/22 individuals with available information), ranging from mild to severe/profound; (b) epilepsy (15/25); (c) neurological impairment, with altered muscle tone (14/22); (d) dysmorphisms (13/20). Nineteen pathogenic KCNT2 variants were found (9 new, 10 reported previously): 16 missense, 1 in-frame deletion of a single amino acid, 1 nonsense, and 1 frameshift. Among tested variants, 8 showed gain-of-function (GoF), and 6 loss-of-function (LoF) features when expressed heterologously in vitro. Quinidine and fluoxetine blocked all GoF variants, whereas loxapine and riluzole activated some LoF variants while blocking others. INTERPRETATION: We expanded the phenotypic and genotypic spectrum of KCNT2-related disorders, highlighting novel genotype-phenotype associations. Pathogenic KCNT2 variants cause GoF or LoF in vitro phenotypes, and each shows a unique pharmacological profile, suggesting the need for in vitro functional and pharmacological investigation to enable targeted therapies based on the molecular phenotype. ANN NEUROL 2023;94:332-349.

An integrated genetic analysis of epileptogenic brain malformed lesions.

Focal cortical dysplasia is the most common malformation during cortical development, sometimes excised by epilepsy surgery and often caused by somatic variants of the mTOR pathway genes. In this study, we performed a genetic analysis of epileptogenic brain malformed lesions from 64 patients with focal cortical dysplasia, hemimegalencephy, brain tumors, or hippocampal sclerosis. Targeted sequencing, whole-exome sequencing, and single nucleotide polymorphism microarray detected four germline and 35 somatic variants, comprising three copy number variants and 36 single nucleotide variants and indels in 37 patients. One of the somatic variants in focal cortical dysplasia type IIB was an in-frame deletion in MTOR, in which only gain-of-function missense variants have been reported. In focal cortical dysplasia type I, somatic variants of MAP2K1 and PTPN11 involved in the RAS/MAPK pathway were detected. The in-frame deletions of MTOR and MAP2K1 in this study resulted in the activation of the mTOR pathway in transiently transfected cells. In addition, the PTPN11 missense variant tended to elongate activation of the mTOR or RAS/MAPK pathway, depending on culture conditions. We demonstrate that epileptogenic brain malformed lesions except for focal cortical dysplasia type II arose from somatic variants of diverse genes but were eventually linked to the mTOR pathway.

Hybrid double-spiral microfluidic chip for RBC-lysis-free enrichment of rare cells from whole blood.

Drug selection and treatment monitoring via minimally invasive liquid biopsy using circulating tumor cells (CTCs) are expected to be realized in the near future. For clinical applications of CTCs, simple, high-throughput, single-step CTC isolation from whole blood without red blood cell (RBC) lysis and centrifugation remains a crucial challenge. In this study, we developed a novel cancer cell separation chip, "hybrid double-spiral chip", that involves the serial combination of two different Dean flow fractionation (DFF) separation modes of half and full Dean cycles, which is the hybrid DFF separation mode for ultra-high-throughput blood processing at high precision and size-resolution separation. The chip allows fast processing of 5 mL whole blood within 30 min without RBC lysis and centrifugation. RBC and white blood cell (WBC) depletion rates of over 99.9% and 99%, respectively, were achieved. The average recovery rate of spiked A549 cancer cells was 87% with as low as 200 cells in 5 mL blood. The device can achieve serial reduction in the number of cells from approximately 1010 cells of whole blood to 108 cells, and subsequently to an order of 106 cells. The developed method can be combined with measurements of all recovered cells using imaging flow cytometry. As proof of concept, CTCs were successfully enriched and enumerated from the blood of metastatic breast cancer patients (N = 10, 1-69 CTCs per 5 mL) and metastatic prostate cancer patients (N = 10, 1-39 CTCs per 5 mL). We believe that the developed method will be beneficial for automated clinical analysis of rare CTCs from whole blood.

Genetic and clinical landscape of childhood cerebellar hypoplasia and atrophy.

PURPOSE: Cerebellar hypoplasia and atrophy (CBHA) in children is an extremely heterogeneous group of disorders, but few comprehensive genetic studies have been reported. Comprehensive genetic analysis of CBHA patients may help differentiating atrophy and hypoplasia and potentially improve their prognostic aspects. METHODS: Patients with CBHA in 176 families were genetically examined using exome sequencing. Patients with disease-causing variants were clinically evaluated. RESULTS: Disease-causing variants were identified in 96 of the 176 families (54.5%). After excluding 6 families, 48 patients from 42 families were categorized as having syndromic associations with CBHA, whereas the remaining 51 patients from 48 families had isolated CBHA. In 51 patients, 26 aberrant genes were identified, of which, 20 (76.9%) caused disease in 1 family each. The most prevalent genes were CACNA1A, ITPR1, and KIF1A. Of the 26 aberrant genes, 21 and 1 were functionally annotated to atrophy and hypoplasia, respectively. CBHA+S was more clinically severe than CBHA-S. Notably, ARG1 and FOLR1 variants were identified in 2 families, leading to medical treatments. CONCLUSION: A wide genetic and clinical diversity of CBHA was revealed through exome sequencing in this cohort, which highlights the importance of comprehensive genetic analyses. Furthermore, molecular-based treatment was available for 2 families.

An integrated high-throughput microfluidic circulatory fluorescence-activated cell sorting system (µ-CFACS) for the enrichment of rare cells.

There is an increasing need for the enrichment of rare cells in the clinical environments of precision medicine, personalized medicine, and regenerative medicine. With the possibility of becoming the next-generation cell sorters, microfluidic fluorescence-activated cell sorting (µ-FACS) devices have been developed to avoid cross-contamination, minimize device footprint, and eliminate bio-aerosols. However, due to highly precise flow control, the achievable throughput of the µ-FACS system is generally lower than the throughput of conventional FACS devices. Here, we report a fully integrated high-throughput microfluidic circulatory fluorescence-activated cell sorting (µ-CFACS) system for the enrichment of clinical rare cells. A microfluidic sorting cartridge has been developed for enriching samples through a sequential sorting process, which was further realized by the integration of both fast amplified piezoelectrically actuated on-chip valves and compact pneumatic cylinders actuated on-chip valves. At an equivalent throughput of ∼8000 events per second (eps), the purity of rare fluorescent microparticles has been significantly increased from ∼0.01% to ∼27.97%. An enrichment of ∼9400-fold from 0.009% to 81.86% has also been demonstrated for isolating fluorescently labelled MCF-7 breast cancer cells from Jurkat cells at an equivalent sorting throughput of ∼6400 eps. With the advantages of high throughput and contamination-free design, the proposed integrated µ-CFACS system provides a new option for the enrichment of clinical rare cells.

Integrated system for detection and molecular characterization of circulating tumor cells.

Circulating tumor cells (CTCs) invade blood vessels in solid tumors and promote metastases by circulating in the blood. CTCs are thus recognized as targets for liquid biopsy and can provide useful information for design of treatments. This diagnostic approach must consider not only the number of CTCs but also their molecular and genetic characteristics. For this purpose, use of devices that enrich CTCs independent of these characteristics and detectors that recognize various CTC characteristics is essential. In the present study, we developed a CTC detection system comprising ClearCell FX and ImageStream Mark II. We clarified the analytical performance of this system by evaluating recovery rate, lower limits of detection, and linearity. These parameters are critical for detecting rare cells, such as CTCs. We tested these parameters using three cell lines with different expression levels of the epithelial marker-epithelial cell adhesion molecule (EpCAM) and spiked these cells into whole-blood samples from healthy donors. The average recovery rate and lower limit of detection were approximately 40% and five cells/7.5 mL of whole blood, respectively. High linearity was observed for all evaluated samples. We also evaluated the ability of the system to distinguish between normal and abnormal cells based on protein expression levels and gene amplification and found that the system can identify abnormal cells using these characteristics. The CTC detection system thus displays the ability to distinguish specific characteristics of CTC, thereby providing valuable information for cancer treatment.

KCNT1-positive epilepsy of infancy with migrating focal seizures successfully treated with nonnarcotic antitussive drugs after treatment failure with quinidine: A case report.

BACKGROUND: Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the early-onset epileptic encephalopathies resistant to antiepileptic drugs, therefore carrying an extremely poor neurodevelopmental outcome. KCNT1, encoding for a sodium-activated potassium channel (KCa4.1 channel), has recently been reported as the major gene responsible for EIMFS. Since gain of function is the only type of mutation identified in patients with EIMFS, quinidine, a partial antagonist of KCa4.1 channel, is considered as a potential candidate for targeted treatment of EIMFS. However, treatment results reported so far vary from seizure-free state to no response, and cardiac side effect remains a challenge for dose titration and long-term treatment. CASE REPORT: Our case was an infant diagnosed with EIMFS with confirmed mutation in KCNT1 gene. Quinidine therapy was started as early as 9 months old. Within the first month of treatment, the number of seizures reduced to about one third. However, seizure-free state was not obtained and his neuropsychological development remained severely delayed. After 16 months of treatment, quinidine had to be discontinued because of cardiac side effects. At 27 months of age, however, his seizures suddenly stopped and he remained seizure-free for five days. This coincided with the prescription of tipepidine, a commonly used antitussive, administered for his persistent cough. Reduction in seizure frequency was also observed with dextromethorphan, another conventional antitussive drug. Although the relation between these treatments and his symptom improvement is a matter of elucidation, there is a possibility that these nonnarcotic antitussive drugs might play a role in the treatment of EIFMS.

De novo PHACTR1 mutations in West syndrome and their pathophysiological effects.

Trio-based whole exome sequencing identified two de novo heterozygous missense mutations [c.1449T > C/p.(Leu500Pro) and c.1436A > T/p.(Asn479Ile)] in PHACTR1, encoding a molecule critical for the regulation of protein phosphatase 1 (PP1) and the actin cytoskeleton, in unrelated Japanese individuals with West syndrome (infantile spasms with intellectual disability). We then examined the role of Phactr1 in the development of mouse cerebral cortex and the pathophysiological significance of these two mutations and others [c.1561C > T/p.(Arg521Cys) and c.1553T > A/p.(Ile518Asn)], which had been reported in undiagnosed patients with intellectual disability. Immunoprecipitation analyses revealed that actin-binding activity of PHACTR1 was impaired by the p.Leu500Pro, p.Asn479Ile and p.Ile518Asn mutations while the p.Arg521Cys mutation exhibited impaired binding to PP1. Acute knockdown of mouse Phactr1 using in utero electroporation caused defects in cortical neuron migration during corticogenesis, which were rescued by an RNAi-resistant PHACTR1 but not by the four mutants. Experiments using knockdown combined with expression mutants, aimed to mimic the effects of the heterozygous mutations under conditions of haploinsufficiency, suggested a dominant negative effect of the mutant allele. As for dendritic development in vivo, only the p.Arg521Cys mutant was determined to have dominant negative effects, because the three other mutants appeared to be degraded with these experimental conditions. Electrophysiological analyses revealed abnormal synaptic properties in Phactr1-deficient excitatory cortical neurons. Our data show that the PHACTR1 mutations may cause morphological and functional defects in cortical neurons during brain development, which is likely to be related to the pathophysiology of West syndrome and other neurodevelopmental disorders.

A patient with peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome, and severe hypoganglionosis associated with a novel SOX10 mutation.

In this report, we present the case of a female infant with peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome, and Hirschsprung disease (PCWH) associated with a novel frameshift mutation (c.842dupT) in exon 5, the last exon of SOX10. She had severe hypoganglionosis in the small intestine and entire colon, and suffered from frequent enterocolitis. The persistence of ganglion cells made both the diagnosis and treatment difficult in the neonatal period. She also showed hypopigmentation of the irises, hair and skin, bilateral sensorineural deafness with hypoplastic inner year, severe demyelinating neuropathy with hypotonia, and diffuse brain hypomyelination. The p.Ser282GlnfsTer12 mutation presumably escapes from nonsense-mediated decay and may generate a dominant-negative effect. We suggest that hypoganglionosis can be a variant intestinal manifestation associated with PCWH and that hypoganglionosis and aganglionosis may share the same pathoetiological mechanism mediated by SOX10 mutations.

Total Capture, Convection-Limited Nanofluidic Immunoassays Exhibiting Nanoconfinement Effects.

Understanding nanoconfinement phenomena is necessary to develop nanofluidic technology platforms. One example of nanoconfinement phenomena is shifts in reaction equilibria toward reaction products in nanoconfined systems, which have been predicted theoretically and observed experimentally in DNA hybridization. Here we demonstrate a convection-limited nanofluidic immunoassay that achieves total capture of a target analyte and an apparent shift in the antibody-antigen reaction equilibrium due to nanoconfinement. The system exhibits wavefronts of the target analyte that propagate along the length of the nanochannel at a velocity much slower than that of the carrier fluid. We apply an analytical model describing the propagation of these wavefronts to determine the density of capture antibody binding sites in the enclosed nanochannel for a known concentration of the target analyte. We then use this binding site density to estimate the concentration of solutions with 5× and 10× less analyte. Our analysis suggests that nanoconfinement results in a preference toward binding of the target analyte with the surface-grafted capture antibody, as evidenced by an apparent reduction in the equilibrium dissociation constant. Our findings motivate the advancement of new biomedical and chemical synthesis technologies by leveraging nanoconfinement effects, and demonstrate a useful platform for studying the effect of nanoconfinement on chemical systems.

A single-molecule ELISA device utilizing nanofluidics.

Single molecule analysis is desired in many areas that require the analysis of ultra-small volume and/or extremely low concentration samples (e.g., single-cell biology, medicine diagnosis, virus detection, etc.). Due to the ultra-small volume or concentration, the sample contains only single or countable analyte molecules. Thus, specific single molecules should be precisely processed and detected for analysis. However, except nucleic acids, most molecules are difficult to amplify, and a new analytical methodology for specific single molecules is thus essential. For this, efficient chemical processing and detection, which are important analytical elements, should be developed. Here, we report a single-molecule ELISA (enzyme-linked immunosorbent assay) device utilizing micro/nanofluidic technology. Both chemical processing and detection were integrated into an ultra-small space (102 nm in size), and the integration allowed precise processing (∼100% capture) and detection of a specific single molecule (protein) for the first time. This new concept and enabling technology represent a significant innovation in analytical chemistry and will have a large impact on general biology and medicine.

Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis.

PURPOSE: To clarify the relationship between macrocephaly and neurodevelopmental disorders, as well as identify the prevalence of PTEN mutations in autism spectrum disorders with macrocephaly in Japan. SUBJECTS AND METHODS: Diagnostic and other medical information of children with macrocephaly younger than 4years (n=93) were collected for analysis. PTEN gene mutation analysis was conducted in another set of 16 macrocephalic individuals aged 3-22years. RESULTS: Sixteen macrocephalic children were associated with neurodevelopmental disorders, including autism spectrum disorders (ASDs) (n=6), autistic traits (n=5), intellectual disability (n=5), attention deficit hyperactivity disorder (n=1), developmental coordination disorders (n=1), and language disorder (n=1). Male gender was significantly linked to these disorders, whereas a family history and degree of macrocephaly were not significantly linked to the diagnosis. A novel mutation in the PTEN gene was identified in a 16-year-old girl with autism, mental retardation, language delay, extreme macrocephaly (+4.7SD) with a prominent forehead, and digital minor anomalies. CONCLUSION: Children with macrocephaly, particularly males, are at a higher risk of neurodevelopmental disorders, rather than progressive etiologies, such as hydrocephalus and neurodegenerative disorders. The data provide a basis for routine health checks for young children in Japan, including the follow-up management and possible screening of PTEN mutations in children with ASDs and macrocephaly.

A patient with early myoclonic encephalopathy (EME) with a de novo KCNQ2 mutation.

BACKGROUND: The potassium voltage-gated channel subfamily Q member 2 (KCNQ2) gene has been reported to be associated with various types of epilepsy, including benign familial neonatal seizure (BFNS), early infantile epileptic encephalopathy (EIEE), and unclassified early onset encephalopathies. We herein report a patient with early myoclonic encephalopathy (EME) caused by a KCNQ2 mutation. CASE REPORT: A male infant started to exhibit erratic myoclonus several days after birth and apnea attacks lasting for seconds with desaturation. One month after birth, his myoclonuses worsened in frequency. Electroencephalogram (EEG) showed a burst and suppression pattern, and myoclonuses occurred in the burst phase with diffuse polyspikes on EEG. At five months, inter-ictal EEG revealed hypsarrhythmia, but his attacks were still only myoclonuses. ACTH treatment was effective and the myoclonus frequency markedly decreased. At one year of age, whole-exome sequencing revealed a heterozygous mutation of the KCNQ2 gene (NM_172107.2): c.601C>T; p.(Arg201Cys), which was confirmed as de novo by Sanger sequencing. This mutation lies within the extracellular portion of the S4 voltage sensor. CONCLUSION: Most patients with a KCNQ2 mutation present with seizures starting in the neonatal period with varying severity, ranging from BFNS to Ohtahara syndrome. Furthermore, KCNQ2 appears to be a causative gene for EME.

An Xq22.1q22.2 nullisomy in a male patient with severe neurological impairment.

The proteolipid protein 1 gene (PLP1) is located on chromosome Xq22.2 and is related to X-linked recessive leukoencephalopathy (Pelizaeus-Merzbacher disease: PMD). Compared to PLP1 duplications, which are a major contributor to PMD, chromosomal deletions in this region are rare and only a few PMD patients with small deletions have been reported, suggesting that large deletions of this region would cause embryonic lethality. Previously, we have reported female patients, with chromosomal deletions in this region, who showed severe developmental delays and behavioral abnormalities. In this study, we identified the first case of a male patient associated with an Xq22 nullisomy in a region proximal to PLP1. The patient showed severe neurological impairment and was bedridden. Brain magnetic resonance imaging revealed a severely reduced cerebral volume. The chromosomal region proximal to PLP1 was considered to be significantly important for brain development.

Droplet-Free Digital Enzyme-Linked Immunosorbent Assay Based on a Tyramide Signal Amplification System.

Digital enzyme-linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. This study presents a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In our method, tyramide substrate reacts with horseradish peroxidase (HRP) labeled with an immunocomplex on beads, and the substrate is converted into short-lived radical intermediates. By adjusting the bead concentration in the HRP-tyramide reaction and conducting the reaction using freely moving beads, tyramide radicals are deposited only on beads labeled with HRP and there is no diffusion to other beads. Consequently, the fluorescence signal is localized on a portion of the beads, making it possible to count the number of labeled beads digitally. The performance of our method was demonstrated by detecting hepatitis B surface antigen with a limit of detection of 0.09 mIU/mL (139 aM) and a dynamic range of over 4 orders of magnitude. The obtained limit of detection represents a >20-fold higher sensitivity than conventional ELISA. Our method has potential applications in simple in vitro diagnostic systems for detecting ultralow concentrations of protein biomarkers.

Stimulus-induced reflex epileptic spasms in 5p- syndrome.

Here we describe two patients with 5p- syndrome who suffered from epilepsy characterised by stimulus-induced epileptic spasms manifesting as head nodding. In patient 1, a series of spasms were exclusively triggered by eating, and were associated with diffuse high-voltage slow waves on ictal EEG, particularly presenting as a positive slow potential at the left mid-temporal area. Clusters of sharp waves with negative polarity emerged in the same area during the inter-spasm periods during eating. In patient 2, spasms were provoked by either eating or micturition. Ictal EEG of clustered spasms after micturition showed positive slow or triphasic waves, which correlated with each spasm, over the bifrontal and vertex areas. These findings suggest that the focal cortical areas act as trigger regions in reflex epilepsies, and that a spasm-generator responsible for the execution of reflex spasms exists either in other cortical areas or in the subcortical structures. Although epilepsy is an unusual complication of 5p- syndrome, this syndrome may have a propensity to develop reflex epilepsy, particularly epileptic spasms. However, identification of responsible genes and their roles in this phenotype requires further investigations.

Risk factors for acute pancreatitis in patients with severe motor and intellectual disabilities.

BACKGROUND: Acute pancreatitis in patients with severe motor and intellectual disability (SMID) is a rare but life-threatening condition. Possible causes of acute pancreatitis in these patients including valproic acid therapy, hypothermia and nasoduodenal tube feeding, have not yet been investigated in detail. The aim of this study was therefore to investigate the risk factors for acute pancreatitis in patients with SMID. METHODS: Five SMID patients with acute pancreatitis and 15 SMID patients without acute pancreatitis were reviewed. Age; serum total cholesterol, triglyceride, total protein, and albumin; height; bodyweight; body surface area; body mass index; daily calorie intake; daily calorie intake per unit of body mass surface area; daily calorie intake per kilogram bodyweight; and valproic acid usage were examined. RESULTS: A statistically significant difference was observed in serum albumin level between the two groups (P = 0.026). CONCLUSION: The mechanism of acute pancreatitis in these patients was considered as pancreatic morphological change, acinar damage, and elevated serum trypsinogen level caused by malnutrition. It is likely that acute pancreatitis in patients with SMID occurs due to the same mechanism as in anorexia nervosa and malnourished patients. To prevent acute pancreatitis in these patients, it is important to maintain adequate nutritional status.

Extended nanofluidic immunochemical reaction with femtoliter sample volumes.

The growing need to optimize immunoassay performance driven by interest in analyzing individual cells has resulted in a decrease in the amount of sample required. Miniaturized immunoassays that use ultra-small femtoliter to attoliter sample volumes, a range known as the extended nanospace, can satisfy this analytical need; however, capturing every targeted molecule without loss in extended nanochannels for subsequent detection remains challenging. This is the first report of a successful extended nanofluidics-based quantitative immunochemical reaction capable of high capture efficiency using a femtoliter-scale sample volume. A novel patterning method using a photolithographic technique with vacuum ultraviolet light and low-temperature (100 °C) bonding enables patterning of functional groups for antibody immobilization before bonding, resulting in an immunochemical reaction space of only 86 fL. Reaction rate analyses indicate a decrease in the required sample volume to 810 fL and improvement in the limit of detection to 3 zmol, 5-6 orders of magnitude better than possible with the microfluidic immunoassay format. Highly efficient (near 100%) immunochemical reactions on a seconds time scale are possible due to the nm-scale diffusion length, which should be advantageous for the analysis of ultra-low-volume samples.

Double recurrence of group B streptococcus bacteremia in an immunocompetent infant.

We report an immunocompetent infantile case wherein group B streptococcus bacteremia recurred two times. The isolated bacteria which colonized in his nasopharyngeal cavity might be a source of repetitive infection. Although the best action has not been established yet, penicillin G as the prophylaxis seemed to be effective in this case.